KNOW-HOW LICENSE AND STOCK PURCHASE AGREEMENT

THIS AGREEMENT is made and entered into as of the day it has been signed by both parties below

by and between:

Regenicin, Inc., having a principal address at 10 High Court, Little Falls, NJ 07424 (hereafter Regenicin); and Lonza Walkersville, Inc., having a principal address at 8830 Biggs Ford Road, Walkersville, MD 21793 (hereafter LWI or Lonza);

INDEX OF ARTICLES

1 DEFINITIONS

2 OBLIGATIONS OF THE PARTIES

3 KNOW-HOW

4 GRANT

5 PATENT RIGHTS

6 PAYMENTS

7 MANUFACTURING

8 CLINICAL TRIALS

9 STOCK PURCHASE AGREEMENT

10 DISTRIBUTION

11 PAYMENTS

12 CONFIDENTIALITY

13 TERM AND TERMINATION

14 INFRINGEMENT AND INDEMNITY

15 MISCELLANEOUS

INDEX OF EXHIBITS

Exhibit A - List of Patent Rights

Exhibit B - Hourly rate for calculation of cost of Future Know-How

Exhibit C – AFIRM Grant Application

Exhibit D - The Amended and Restated Exclusive License Agreement between The Regents of the University of California and Cutanogen Corporation.

Exhibit E – The First Amendment to the Amended and Restated Exclusive License Agreement between The Regents of the University of California and Cutanogen Corporation.

Exhibit F - The License Agreement between Cutanogen Corporation on the one hand and the University of Cincinnati and Shriners Hospitals for Children on the other hand.

Exhibit G – Amendment to the License Agreement between Cutanogen Corporation on the one hand and the University of Cincinnati and Shriners Hospitals for Children on the other hand..

Exhibit H - Settlement Agreement and Release dated February 2, 2006 between the Shriners Hospital for Children, Cutanogen Corporation, the Shareholders of Cutanogen Corporation, and Cambrex Bio Science Walkersville, Inc.

Exhibit I - Manufacturing Agreement.

Exhibit J - Stock Purchase Agreement.

WHEREAS, LWI owns all of the issued and outstanding capital stock of Cutanogen Corporation;

WHEREAS, LWI is engaged in the research and development of products used in the life sciences industry, including engineered skin substitute called PermaDerm™;

WHEREAS, Regenicin wishes to assume responsibility for developing PermaDerm™, and to purchase the outstanding capital stock of Cutanogen Corporation;

NOW, THEREFORE, in consideration of the promises and of the mutual covenants and agreements herein set forth, the parties hereto agree as follows:

Article 1. Definitions

1.1           "Affiliate" means any corporation, company or other entity which directly or indirectly controls, is controlled by, or is under common control with, LWI or Regenicin. For the purpose of this definition, the word "control" shall mean the direct or indirect ownership of more than fifty percent (50%) of the outstanding voting stock of the corporation, company, or other entity.

1.2           "Contract Product" means the autologous engineered skin supplement known as PermaDerm™.

1.3           “Current Know-How” means Know-How that is in the possession of LWI, and that exists on the effective date of this Agreement.

1.4           “Future Know-How” means Know-How that is developed after the effective date of this Agreement by LWI at the reasonable request of Regenicin.

1.5           “Know-How” means information in support of a clinical trial for the Contract Product, including, without limitation, information relating to product specifications, manufacturing, testing, facilities, etc. Know-How includes, but is not limited to, Master Batch Records and SOP’s.

1.6           “Patent Rights" shall mean all patents and patent applications in any country or region of the world that cover Contract Product, and that are owned by or are licensed to, LWI including, without limitation, the patents and patent applications that were licensed by LWI from the University of California, and from the University of Cincinnati and the Shriners Hospital for Children. A partial list of Patent Rights is set forth in Exhibit A.

Article 2. Obligations of the Parties

2.1 This Agreement shall only become effective upon Lonza’s receipt of payment in full to be made by Regenicin of the amount due under paragraph 6.1 below (the “Effective Date”).

2.2 During the term of this Agreement, the obligations of LWI shall include, without limitation:

a. produce and transfer Future Know-How to Regenicin in accordance with paragraph 3.2 below;

b. monitor prosecution and maintenance of Patent Rights in accordance with paragraph 5.2 below;

c. maintain the Licenses, as defined below; and

d. execute the Stock Purchase Agreement in accordance with Article 9.1 below.

2.3 During the term of this Agreement, the obligations of Regenicin shall include, without limitation:

a. reimburse LWI for transferring Current Know-How in accordance with paragraph 6.1 below

b. conduct pre-clinical and clinical trials in accordance with paragraph 8.1 below;

c. apply for and obtain approval from the FDA for commercial sales of Licensed Product in accordance with paragraph 8.1 below;

d. reimburse LWI for providing Future Know-How in accordance with paragraph 3.3 below;

e. reimburse LWI for monitoring prosecution of Patent Rights in accordance with paragraph 5.6 below;

f. reimburse LWI for maintaining the Licenses in according with paragraph 5.7 below; and

g. execute the Stock Purchase Agreement in accordance with Article 9.1 below.

h. Pay the purchase price for the outstanding capital stock of Cutanogen Corporation in according with paragraph 6.2 below.

Article 3. Know-How

3.1 Regenicin acknowledges LWI has transferred the Current Know-How.

3.2 Upon the reasonable request of Regenicin, LWI shall produce and transfer to Regenicin Future Know-How.

3.3 LWI shall send invoices to Regenicin for expenses related to the production and transfer to Regenicin of Future Know-How at a rate based on the number of hours spent by employees of LWI in accordance with the rate provided in Exhibit B, which may be updated from time to time by Lonza.

Article 4. Grant

4.1 LWI grants to Regenicin, and Regenicin accepts, an exclusive license to use Know-How for the sole purposes of seeking FDA approval and, upon such approval, to use, market, offer for sale, sell and otherwise dispose of the Contract Product on a worldwide basis, subject to the terms of this Agreement. Notwithstanding the foregoing, to the extent that any grant of rights to Regenicin hereunder would constitute a transfer, assignment or sublicense of Lonza’s or Cutanogen Corporation’s right, interest or title in intellectual property that would trigger any milestone payments under the Stock Purchase Agreement, dated December 2005, by and among Lonza, Cutanogen Corporation and the shareholders of Cutanogen Corporation, then such portion of such grant shall be of no force and effect.

4.2 No licenses or rights, including, without limitation, sublicenses under the Licenses, other than those explicitly granted hereunder are, or shall be deemed to have been, granted under this agreement.

4.3 LWI applied for a grant from the Armed Forces Institute of Regenerative Medicine relating to the Contract Product on March 3, 2009 (hereafter AFIRM Grant Application, attached hereto as Exhibit C). Any money received by Lonza during the term of this agreement as a result of the AFIRM Grant Application shall be applied by LWI to development of the Contract Product in compliance with the conditions of the grant, and the results thereof transferred to Regenicin to the extent permitted under the grant.

4.4 LWI applied for a grant from the Department of Defense relating to the Contract Product (hereafter DoD Grant Application). Any money received by Lonza during the term of this agreement as a result of the DoD Grant Application or any other grant relating to the Contract Product shall be applied by LWI to development of the Contract Product in compliance with the conditions of such grant, and the results thereof transferred to Regenicin to the extent permitted under such grant.

4.5 With respect to the AFIRM grant and any other grant that Lonza or its affiliates receives relating to the Contract Product, including, but not limited to, any grant from the Department of Defense, Lonza or its affiliate(s), as the case may be, shall control all such grants and shall be the only point of contact or interface with the grantor. Regenicin shall not be permitted to communicate, directly or indirectly, with the grantor without the express written permission of Lonza.

Article 5. Patent Rights

5.1 The parties acknowledge that certain Patent Rights relating to the Contract Product were licensed to LWI in the following agreements (hereafter, collectively, the Licenses):

Amended and Restated Exclusive License Agreement between The Regents of the University of California and Cutanogen Corporation for “Living Human Skin Replacements and Cultured Skin Substitutes” effective March 3, 2001 (hereafter California Agreement, attached hereto as Exhibit D;

First Amendment to the Amended and Restated Exclusive License Agreement between The Regents of the University of California and Cutanogen Corporation effective November 23, 2005 (hereafter California Amendment, attached hereto as Exhibit E;

License Agreement between Cutanogen Corporation on the one hand and the University of Cincinnati and Shriners Hospitals for Children on the other hand, effective as of August 24, 1998 (hereafter Cincinnati/SHC agreement), attached hereto as Exhibit F; and

Amendment to License Agreement between Cutanogen Corporation on the one hand and the University of Cincinnati and Shriners Hospitals for Children on the other hand, effective as of December 29, 2005 (hereafter Cincinnati/SHC amendment), attached hereto as Exhibit G.

5.2 LWI shall continue to monitor the prosecution and maintenance of Patent Rights that were licensed to LWI under the Licenses, and shall use its reasonable best efforts to obtain effective patent protection for the Contract Product under Patent Rights licensed thereunder.

5.3 Upon execution of the Stock Purchase Agreement in accordance with paragraph 9.1 below, the California Agreement and the California Amendment will be assigned to Regenicin, subject to the written consent of the University of California in accordance with paragraph 22 of the California Agreement.

5.4 Upon execution of the Stock Purchase Agreement in accordance with paragraph 9.1 below, the Cincinnati/SHC Agreement and the Cincinnati/SHC Amendment will be assigned to Regenicin, subject to the prior consent of the University of Cincinnati and Shriners Hospitals for Children in accordance with paragraph 14.4 of the Cincinnati/SHC Agreement.

5.5 Following the execution of the Stock Purchase Agreement in accordance with paragraph 9.1 below, LWI will use reasonable efforts to obtain, at Regenicin’s sole expense, any consent required as a condition to the assignment of the California Agreement, the California Amendment, the Cincinnati/SHC Agreement and the Cincinnati/SHC Amendment.

5.6 LWI shall send invoices to Regenicin for all expenses related to the prosecution and maintenance of Patent Rights.

5.7 LWI shall send invoices to Regenicin for all expenses related to the maintenance of the Licenses, including, without limitation, the license maintenance fee of Article 6 of the California Agreement.

Article 6. Payments

6.1 Regenicin shall pay to LWI $3,000,000 (three million dollars) as consideration for Current Know-How and other value transferred or to be transferred by Lonza to Regenicin.

6.2 Regenicin shall pay to LWI $2,000,000 (two million dollars) upon approval by the United States Food and Drug Administration of the commercial sale of the Contract Product as the purchase price of the outstanding capital stock of Cutanogen Corporation transferred to Regenicin under the Stock Purchase Agreement in accordance with paragraph 9.1 below.

6.3 Regenicin shall pay to LWI 33% (thirty three percent) of any money received by Lonza or its affiliates during the term of this Agreement as a result of the AFIRM grant application in consideration for the development of the Contract Product conducted by LWI, and the transfer of the results thereof to Regenicin in accordance with paragraph 4.3 above.

6.4 Regenicin shall pay to LWI 33% (thirty three percent) of any money received by Lonza or its affiliates during the term of this Agreement as a result of any other grant application(s), including, but not limited to, as a result of any Department of Defense grant application, in consideration for the development of the Contract Product to be conducted by LWI, and the results thereof to be transferred to Regenicin in accordance with paragraph 4.4 above, with such payment to be made upon receipt of such grant.

6.5 Regenicin shall be responsible for any milestone payments that are owed, whether upon execution of the Stock Purchase Agreement in accordance with paragraph 9.1 below or otherwise, as well as any milestone payments that, but for this Agreement, would not have been owed by LWI to one or more, including all, former owners of Cutanagen Corporation, including, without limitation, Stephen Boyce, the University of Cincinnati and the Shriners Hospital for Children. Milestone payments covered under this paragraph 6.5 include, without limitation, any milestone payments would have been owed under the Settlement Agreement and Release dated February 2, 2006 between the Shriners Hospital for Children, Cutanogen Corporation, the Shareholders of Cutanogen Corporation, and Cambrex Bio Science Walkersville, Inc. (Exhibit H).

Article 7. MANUFACTURING

7.1 During the term of this Agreement and following execution of the Stock Purchase Agreement in accordance with paragraph 9.1 below, LWI will retain the exclusive right to manufacture Contract Product at a customary margin level as more definitively set forth in the Manufacturing Agreement attached hereto as Exhibit I. Regenicin shall order amounts of Contract Product from LWI for clinical trial, and LWI shall manufacture Contract Product in accordance with the Manufacturing Agreement.

7.2 Regenicin shall pick up the Contract Product manufactured by LWI in accordance with Paragraph 7.1 above, and shall arrange for transportation from a facility designated by LWI to a facility designated by Regenicin for each delivery of Contract Product. All costs, taxes and other expenses relating to such delivery and transport, including, without limitation, insurance premiums, shall be at Regenicin's expense.

7.3 Title, and risk of loss or damage, to any shipment of Contract Product shall belong to Regenicin upon pick up of Contract Product by Regenicin in accordance with Paragraph 7.2 above.

7.4 If, at any time following execution of the Stock Purchase Agreement in accordance with paragraph 9.1 below, LWI is, for any reason, unable or unwilling to supply Regenicin’s reasonable requirements for commercial supply of Contract Product and notifies Regenicin to that effect, LWI agrees that Regenicin may then have the Contract Product manufactured by one or more third parties to the extent necessary in order to permit Regenicin to obtain supply of its requirements of Contract Product (and the license grant set forth in paragraph 4.1 above shall be deemed to be modified to the extent necessary for such purpose).

Article 8. Clinical Trials

8.1. During the term of this Agreement, Regenicin shall prepare for and conduct pre-clinical and clinical trials. Regenicin shall use its best efforts during such trials to obtain approval from the FDA for the commercial sale of Contract Product.

8.2 LWI will support the pre-clinical and clinical trials conducted by Regenicin in accordance with paragraph 8.1 above by providing Current Know-How and Future Know-How in accordance with Article 3 above.

Article 9. Stock Purchase Agreement

9.1 Upon payment by Regenicin to LWI of the milestone payments provided for in paragraphs 6.2 and 6.5 above, Regenicin and LWI shall execute the Stock Purchase Agreement attached hereto as Exhibit J. For the sake of clarity, the Stock Purchase Agreement shall not be effective until all of the milestones due under paragraphs 6.2 and 6.5 above are paid in full.

Article 10. Distribution

10.1 After execution of the Stock Purchase Agreement, LWI will retain exclusive distribution rights for the sale of the collagen sponge of the Contract Product to a third party in accordance with a Distribution Agreement to be negotiated by the parties in good faith. Under the Distribution Agreement, LWI will keep 15% of the sale price as a logistics/distribution fee. Any remaining profit from the sale of the collagen sponge will be split equally between the parties.

10.2 The Distribution Agreement will contain a grant by Regenicin to LWI of a worldwide, transferable, non-revocable license under intellectual property owned by or licensed to Regenicin enabling LWI to satisfy its obligations to manufacture Contract Product under paragraph 7.1 above and to distribute Contract Product under paragraph 10.1 above.

Article 11. Payments

11.1 Except for payment under paragraph 6.1, which is due concurrently with the execution hereof, all payments due in accordance with this agreement shall be made in United States dollars within thirty (30) days of the due date or, as the case may be, of the invoice date. Invoices shall be sent to Regenicin and payments shall be made to LWI at the addresses provided in paragraph 15.5 below.

Article 12. Confidentiality

12.1 Each party acknowledges and agrees that the other party (hereinafter the "Donor Party") owns certain confidential information regarding the Contract Product.

12.2 Confidential Information shall mean all information regarding the Contract Product that is hereafter received by a party to this Agreement (hereinafter the "Receiving Party") from the Donor Party, except that which:

(a) was in the public domain prior to the receipt under this Agreement, or thereafter becomes part of the public domain through no fault of the Receiving Party; or

(b) the Receiving Party can show, by credible written records, was in its possession at the time of receipt under this Agreement; or

(c) is received by the Receiving Party from a third party that is not under an obligation to the Donor Party to maintain the information in confidence.

12.3 All Confidential Information received under this Agreement shall be maintained by the Receiving Party in confidence and shall not be disclosed to any other person or entity without prior written approval of the Donor Party, except as is necessary for the Receiving Party to carry out its obligations under this Agreement.

12.4 All physical material containing Confidential Information shall be returned to the Donor Party prior to or immediately upon any termination of this Agreement, provided however, that the Receiving Party may retain one copy of written materials containing Confidential Information strictly for use as a record of information disclosed by the Donor Party, and to be used for no other purpose without the Donor Party's express written consent.

12.5 The Receiving Party hereby indemnifies and holds harmless the Donor Party against any loss resulting from unauthorized disclosure or use of the Confidential Information by the Receiving Party, its agents, or others to whom the Confidential Information has been disclosed by the Receiving Party pursuant to this Agreement.

Article 13. Term And Termination

13.1 Unless otherwise terminated as provided hereunder, this Agreement shall become effective in accordance with paragraph 2.1 and remain in force from its Effective Date until execution of the Stock Purchase Agreement in accordance with Article 9.1 above.

13.2 In the event either party fails or refuses to perform any of its obligations hereunder (hereafter the Defaulting Party), the other party (hereafter the Terminating Party) may, without waiving any other rights, terminate this agreement.

13.3 If the Terminating Party wishes to terminate this agreement in accordance with paragraph 13.2 above, the Terminating Party shall first provide the Defaulting Party with written notice specifying the particulars of such failure or refusal. This Agreement shall terminate thirty (30) days after receipt of such notice by the Defaulting Party unless, within such thirty (30) day period, the default is fully remedied. Notwithstanding the foregoing, this Agreement may be terminated immediately (i) by the non-Defaulting Party in the event the Defaulting Party breaches paragraph 12 above or (ii) by Lonza in the event Regenicin breaches paragraph 11.1.

13.4 Termination of this Agreement shall not relieve either party to this Agreement of its obligations to make any payment that accrue hereunder prior to such termination, or of its obligations of confidentiality in accordance with Article 12 above.

13.5 In the event this Agreement is terminated earlier than provided for in paragraph 13.1 above by either party for any reason, any money transferred to LWI by Regenicin in accordance with Article 6 above shall be non-refundable.

13.6 In the event this Agreement is terminated earlier than provided for in paragraph 13.1 above by either party for any reason, Regenicin shall return all Current Know-How and Future Know-How to LWI, and will not use Current Know-How or Future Know-How for any purpose.

Article 14. Infringement and Indemnity

14.1 LWI represents to Regenicin that, at the time this Agreement is entered into, LWI has no knowledge of any proprietary right that is owned by a third party that would be infringed by Regenicin's sale or use of Contract Product pursuant to this Agreement. However, LWI does not represent or warrant that Regenicin will not be subject to claims by a third party for infringement of a third party's proprietary rights of which LWI is unaware at the time this Agreement.

14.2 Regenicin agrees to hold harmless, defend and indemnify LWI against all damage, claim, expense and liability, including attorney fees, arising in any way from (i) the offering to sell, sale, or use of Contract Product by Regenicin or its agents or (ii) any actions whatsoever taken by Regenicin, its affiliates, or any of its or its affiliates’ respective officers, directors, employees, agents, consultants, independent contractors or representatives in connection with this Agreement, the Contract Product, or Lonza’s (or its affiliates’) relationship with Regenicin.

Article 15. Miscellaneous

15.1 This Agreement shall be governed by and construed in accordance with the laws of The State of New York, without regard to its conflicts of laws rules.

15.2 This Agreement may only be amended in writing signed by both parties. There are no other understandings, agreements or representations, express or implied, not specified herein. If any provision of this Agreement is held to be unenforceable, such provision shall not render this Agreement or any other provision thereof unenforceable. In the event a provision of this Agreement is held to be unenforceable, the parties shall negotiate in good faith so that such unenforceable provision may be replaced by another provision of similar, but enforceable, effect. If such a replacement is not possible or cannot be agreed upon, the parties will negotiate in good faith so that the value of this Agreement to both parties remains the same despite the loss of the provision held to be unenforceable.

15.3 Regenicin shall not use the name Lonza or LWI or the trade name of a Lonza or LWI product without the express written consent of LWI.

15.4 This Agreement shall not be assigned by either party to this Agreement to a third party without the written consent of the other party to this Agreement, such consent not to be unreasonably withheld.

15.5 Any notice required to be given under this Agreement shall be in writing, and shall be deemed to have been received on the day the notice has been transmitted by facsimile to the correct facsimile number, or three days after deposit in the mail, postage prepaid for first class mail, whichever occurs first. All mail shall be addressed as follows:

[remainder of page intentionally left blank]

Exhibit A – List of Patent Rights

Exhibit B – Hourly Rate for calculation of cost of Future Know-How

Lonza

Lonza Cell Therapy – 2009 Fee Schedules

Labor

Other Fees

CONFIDENTIAL

Lonza

Facilities

*There is a minimum booking requirement of 1 week in order to transition projects in and out of production suites.

Storage Rates

Post Production Storage of Product: LWI will store in-process, quarantine, and released product for the client in a validated, monitored, controlled Refrigerator, Freezer (<-20 °C), Ultra Low Freezer (<-70 °C) or LN2 freezer at the cost outlined below:

Sample Storage Fee (based on total number of samples stored)

LN2 Storage: LWI imposes an additional recurring monthly fee for LN2 freezer unit maintenance and storage of samples. These fees are dependent upon the ownership of the LN2 freezer unit:

Storage rates take effect at the beginning of the month following completion of production. Rates are then prorated upon removal of product from storage.

CONFIDENTIAL

Lonza

Shipping Rates

Post Production Shipment of Product: LWI will ship in-process, quarantine, or released product

for a shipping fee plus freight. Client may designate carrier and may use their account number

for direct billing of freight charges. LWI shipping fees are outlined below:

*Freight is charged for both outbound and return shipments if the container is reusable.

Testing Rates

Note: Other test development and testing is available. Please inquire regarding current rates.

CONFIDENTIAL

Exhibit C – AFIRM Grant Application
Lonza

02 March 2009

Prof. David Devore

Chief Operating Officer

CeMAR and RCC Consortium of AFIRM

NJ Center for Biomaterials

145 Bevier Road

Piscaraway, NJ 08854

Dear Professor Devore:

On behalf of Lonza Walkersville Inc. (LWI) we submit our grant proposal titled “Expedited availability of autologous engineered human skin for treatment of burned soldiers” to the Rutgers – Cleveland Clinic Consortium of the Armed Forces Institute of Regenerative medicine for your consideration.

Please note that the information from LWI contained in this proposal is confidential in nature and describes proprietary facilities, processes and testing. Please treat our confidential information appropriate.

LWI appreciates the opportunity Dr. Kohn provided to us by allowing us to participate in this proposal. We have greatly appreciated the support and guidance provided by the RCCC-AFIRM team durning development of this proposeal. Should the proposeal receive funding, LWI looks forward to working with RCCC-AFIRM to carry out the clinical trial of LWI’s Engineering Skin Substitute in the near future.

Please do not hesitate to contact LWI should you have any questions about our proposal of need additional information.

Sincerely,

	Rutgers - Cleveland Clinic Consortium
Proposal Clinical Trial Supplemental Funding Due: March 3, 2009

Project: Expedited availability of autologous engineered human skin for treatment of burned soldiers

Project Title:

Expedited Availability of Autologous Engineered Skin Substitutes for Treatment of Burned Soldiers

1.1 Overview

This proposed study involves the production of a novel and superior engineered skin substitute for the treatment of patients suffering from extensive, deep burns. The autologous engineered skin offers reductions in morbidity and mortality after life-threatening burns by decreasing dramatically the need for skin grafts to complete wound closure. Secondary benefits of the engineered skin may include, but not be limited to: fewer surgical procedures for skin grafting, improved cosmetic outcomes, and shorter hospital length of stay. To date, these projected advantages have been demonstrated in a clinical trial involving over 50 pediatric patients

1-3. Specifically, the funding being requested for this phase of the program will support the following deliverables:

• Implementation of cGMP manufacturing for the Engineered Skin Substitute “ESS” (trade name, PermaDerm) by Lonza Walkersville, Inc. (LWI) which holds licenses to patents for the platform technologies. LWI has proven capabilities for manufacture and delivery of cell therapies with operational systems for full validation of product purity and safety. (See section 4.3)

• Preparation and submission of an Investigational Device Exemption (IDE) application to the US Food and Drug Administration (FDA) to perform a limited study with ESS in patients with full-thickness burns involving greater that 50% of the Total Body Surface Area (TBSA). (See section 5)

• Performance of an initial clinical trial in 8-10 subjects to demonstrate safety and to prove the principle for reduced morbidity with ESS in comparison to meshed, split-thickness skin grafts. Successful completion of this study is expected to enable performance of a pivotal trial and lead to FDA approval of this advanced therapy. (See section 6)

1.2 Clinical Setting and Unmet Needs. Medical needs. Mortality and morbidity from burns, trauma, and other skin loss injuries remain significant medical and socio-economic problems estimated to cost more than $1 billion annually in treatment costs and lost productivity 4,5. Burns in the civilian population cause more than 900,000 hospital days in the US annually 6,7, and full-thickness burns require treatment by excisional debridement and split-thickness skin grafting. From 2003-2007, the burn unit at Fort Sam Houston (USAISR) had 1497 hospitalizations, including 656 military, of which 540 were related to the conflict in Iraq 8. However, victims of large burns do not have sufficient donor skin to complete grafting without multiple reharvestings of donor sites at 7-10 day intervals. With each harvest, healing time increases as epithelial sources (glands, follicles) are removed, leaving wounds and donor sites susceptible to microbial contamination. Sepsis, which develops in part from microbial contamination and invasion in wounds, accounts for 75% of deaths from burn injuries9, and is often associated with multiple organ failure10. Other major aspects of recovery from burns, including immune function, positive nitrogen balance 11,12 and physical therapy, all depend on completion of wound closure. A significant source of long-term morbidity is development of scar at both the donor sites of skin grafts, and in wounds grafted with meshed and widely-expanded skin grafts. Conversely, it is well-known that grafting of wounds with sheet grafts suppresses scar formation.

1.3 Technology Overview. Autologous Engineered Skin Substitutes (ESS). Over more than 20 years, preclinical and clinical studies have resulted in development of autologous ESS. Classified as a medical devices, ESS currently consists of a lyophilized sponge of collagen and chondroitin-sulfate, populated with cultured dermal fibroblasts and epidermal keratinocytes which organize into an analog of skin tissue (Figure 1). The device develops epidermal barrier and basement membrane 13, and releases high levels of angiogenic growth factors, including but not limited to, Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), and Transforming Growth Factor-beta 1 (TGF-β1) 14-16. In addition, both eratinocytes and fibroblasts in culture are known to release inflammatory mediators which promote transient development of fibro-vascular tissue 17.

Figure 1. Histology of A) native human skin (NHS), and C) engineered skin substitute (ESS). The epidermal anatomy of ESS resembles closely that of NHS with proliferating keratinocytes of the epidermal component (e) attached to the dermal component (d), nucleated suprabasal cells analogous to the spinous layer in NHS, and a keratinized surface layer that resembles the protective stratum corneum. Adhesion of the epidermis results from development of basement membrane proteins, B) collagen IV and D) laminin 5; E) BrdU-positive nuclei; F) Integrin β4. Scale bar = 0.1 mm.

1.4 Technology transfer and commercial development of ESS. Platform technologies for ESS were originated by Steven Boyce, PhD, at the University of Cincinnati and the Shriners Hospitals for Children between 1989-2009, and at the University of California San Diego between 1985-1988. These technologies are covered by five US patents 18-22, and two European patents 23,24 (see section 11). Based on clinical successes with these technologies, they were licensed to Cutanogen Corporation which was founded by Dr. Boyce. A business plan was developed which included alternative pathways to product manufacturing and marketing through the Investigational Device Exemption (IDE)/Pre-Market Approval (PMA), and/or Humanitarian Device Exemption (HDE) mechanisms of regulatory clearance by FDA. In 2006, Cutanogen Corporation was acquired by Cambrex BioScience Walkersville, Inc (CBSW). In 2007, CBSW and Cutanogen were acquired by Lonza Corporation which is a global biopharma company based in Basel, Switzerland, and which reported 2008 revenues of CHF 2.94 billion 25. Subsequently, CBSW changed its name to Lonza Walkersville, Inc. (LWI), but continued operations from its facilities in Walkersville, Maryland. LWI has made a corporate commitment to the field of cell therapy, and has comprehensive facilities for manufacture of cellular therapeutics in full compliance with regulatory standards for current Good Manufacturing Practices (cGMP; see details in section 4.3). LWI has proceeded with technology transfer for ESS, and has completed multiple engineering runs to manufacture PermaDerm branded ESS. With capabilities in formulation of pharmaceutical-grade nutrient media, tissue acquisition, cell and tissue culture, and cell banking, LWI has an established record as a leader in development and delivery of tissue engineered medical products for regenerative medicine.

In this context, ESS remain the most advanced autologous skin grafts that have been tested in extensive clinical trials. As described above, the dermal component promotes development of epidermal barrier, basement membrane, and release of angiogenic factors to stimulate rapid vascularization. ESS can be handled easily, and forms functional skin tissue within two weeks after transplantation allowing early rehabilitation. By four weeks after grafting, pressure garments can be worn without mechanical loss of healed skin. The primary benefits which have been demonstrated in burns involving greater than 50% of the total body surface area (TBSA) are: a) reduction of requirements for harvesting of donor skin to complete closure 1,26; and, b) lower mortality27. Secondary benefits may include fewer surgical procedures for grafting, earlier wound closure, shorter length of hospitalization, and reduced morbidity from scar. A more complete summary of clinical results is shown below in Preliminary Data.

2. Benefits to the patient:

Clinical Need: Prompt and effective wound closure remains a rate-limiting factor in recovery from extensive, deep burn injuries. To address this limitation, autologous engineered skin substitutes (ESS) have been developed and clinically tested as an adjunctive treatment to conventional skin grafting. Completed clinical studies show a reduction in the requirement for harvesting donor skin to complete wound closure. Technology for ESS has been licensed to Lonza Walkersville, Inc. which has completed technology transfer and product development. ESS may allow reductions in morbidity and mortality for soldiers who are casualties of combat-related burn injuries. Stated simply, autologous ESS offer a life-saving alternative therapy to patients with catastrophic burn injuries.

This proposal complements the funded AFIRM project for ESS by expediting the path to clinical treatment of wounded soldiers. This supplement provides precisely the resources needed to facilitate and expedite the availability of ESS for clinical study and treatment of extensive burns in military populations. The requested funding will also serve to establish a platform of clinical studies at the USAISR or other clinical centers to which advanced models of ESS with pigment or vascular networks may proceed more rapidly. Performance of the clinical studies will be sponsored by the Lonza Walkersville, Inc., with full regulatory support from the Clinical Trials Core facility of the RCCC-AFIRM directed by Stanton Gerson, MD, at the Case Western Reserve University.

3. Impacts:

Availability of autologous ESS will reduce morbidity from harvesting of donor skin autografts, and reduce mortality as previously demonstrated in pediatric populations with burns of greater than 50% TBSA 1,26,27. The enrollment criterion of 50% TBSA burns relates to the amount of available donor skin, and 4-week time period required to fabricate the study device. Patients with burns smaller than 50% TBSA can be treated by conventional grafting in about 4 weeks. For massive burns (>50% TBSA), increased availability of engineered skin is also expected to reduce total numbers of surgical procedures for skin grafting, total length of hospital stay intensive care days, total blood loss and requirements for transfusion, and to improve functional outcome and quality of life after discharge from the hospital. However, the full impact of this therapy has been reduced by the limited quantity of devices which can be generated in Dr. Boyce’s research laboratory (i.e., ~900 cm2 or ~1 ft2 each week). Based on an approximate TBSA of an adult male of 2.0 m2, and a demonstrated ability to expand donor skin by 100 times its original area, about 0.02 m2 (200 cm2), or 1% TBSA of uninjured skin is needed to resurface the entire body. This principle for conservation of donor skin will be proven in the proposed study, but on a reduced scale to meet the requirements of statistical power analysis (see section 6.5) and the available budget. An average dosage of study devices will be 0.4m2 (4,000 cm2) generated from a skin biopsy of 40 cm2. This dosage in an adult male represents ~20% of the TBSA. Table 1 shows an example for an adult with a full-thickness burn of 80% TBSA. This example assumes expansion of AG by 1:4, and ESS by 1:100.

Table 1. Theoretical (white middle row) and proposed (shaded bottom row) impacts (shaded columns) of ESS compared to split-thickness skin autograft (AG) for a full-thickness burn of 80% TBSA

The budget of this proposal will provide 40,000 cm2 of ESS for a clinical trial in 8-10 subjects. Therefore, the dose of ESS for each subject will average 4,000 cm2 (4.4 ft2) and be generated from a skin biopsy of an average area of 40 cm2 (0.044 ft2). This design will prove the principle of the primary end point which is reduction of donor skin harvested for wound closure. The full clinical impacts of the ESS technology will require larger doses of ESS per patient which will require commercial scale-up after successful completion of this study. After scale-up, 1 m2 of ESS could be delivered in 4-6 weeks.

4. Preliminary Data:

4.1 Preclinical studies. Characterizations of ESS in vitro have included anatomic and physiologic comparisons with natural human skin, incorporation of melanocytes to generate pigmentation (Figure 2), addition of microvascular endothelial cells to promote organization of a vascular plexus 28, and grafting to athymic mice to assess changes in these properties, and stability of healed skin over extended periods of time 13,29. Genetically-modified ESS have also been reported by Dorothy Supp, Ph.D., for expression of PDGF, VEGF, beta-defensins, both in vitro and in after grafting to athymic mice 30,31. This model has also been used to study electro-spun biopolymer scaffolds and stem cell populations. The model is also readily adaptable for studies of regeneration of nerve and epidermal adnexi (glands and hair).

Figure 2. Density-dependent pigmentation of ESS at 5 weeks after grafting to athymic mice. Upper left) 0 HM added, and no pigment develops. Upper right) 400 HM added to a 4 cm2 ESS graft (1X102 HM/cm2) develop infrequent (<40) foci of pigment. Lower right) HM added at 1X103 HM/cm2 develop partial (~50% area) pigmentation. Lower left) Addition of 1X104 HM/cm2 generates complete pigmentation in 5 weeks.

4.2 Clinical studies of ESS in burns. Boyce et al. 2006. J Trauma Crit Care 60(4):821-829. Engineered skin substitutes (ESS) were evaluated in 40 patients under IDE G980023 between 1998 and 2003. From 2003-2005, an additional 14 patients were evaluated for a total of 54.2 Although ESS remain investigational, they have become part of the local treatment protocol for patients suffering from burns of greater than 50% of the total body surface area (“TBSA”; Fig 3). Data on the following pages describe the impacts of ESS on quantitative coverage of burns, and on qualitative assessment of scar. ESS protocols now conform to clinical schedules for grafting in a two-stage procedure, irrigation with antimicrobials for 5 days, and initiation of rehabilitation at 7-10 days after grafting. The primary benefits to patient recovery are reduction of requirements for donor skin harvesting, and reduction in the mesh ratio for split-thickness skin grafts. These benefits result in fewer donor sites, less pain and reduced scar at both the harvest site and the graft site.

Patients and their families affirm these benefits. Percentages of treated areas closed at post-operative day POD 14, and the ratio of closed to donor areas at POD 28 (54 patients) are shown in Figure 4. Engraftment at POD 14 was 79.5 ± 2.1% for ESS and 95.7 ± 2.0% for skin autograft AG, (Fig 4A). Wounds closed with ESS covered 61.5 ± 8.4 times the area of the donor biopsy (Fig 4B) compared to 4:1 expansion of meshed autograft. These values were different statistically (p=0.006) by one-sample t-test, and demonstrate the reduction of donor skin harvesting by grafting of ESS in place of AG. This result defines a new medical benefit to burn patients by autologous ESS. These data demonstrate that comparable rates of engraftment for ESS and AG, and that 1% TBSA of donor skin can close ~60% TBSA of excised burn. Vancouver scores for ESS were statistically lower (p<0.05) than AG during the first six months after grafting, and not different at six months or after (Fig 4C).

Figure 3. Clinical photos after grafting of engineered skin substitutes (ESS) and meshed, split-thickness skin autograft (AG) to the torso of a patient with 77% TBSA burns. A,B) ESS applied in the operating room; C) Post-operative day (POD) 14, wounds closed & rehab begins; D) POD 69, stable wound closure, no blisters, no regrafting, E) POD 479, pliable, hypopigmented skin. Scales in centimeters.

Figure 4. Quantitative outcomes of ESS and AG in treatment of pediatric burns (n = 54). A) % of treated area closed shows ~80% engraftment of ESS; B) Closed: donor ratio; C) Vancouver scores for ESS were as good or better than AG during the first year after grafting. (*, p<0.05) Importantly, the closed: donor ratio of ~60 (B) demonstrates a significant reduction in donor skin harvesting for patients treated with ESS, and defines a new medical benefit for these patients.

Two recent cases of pediatric burns of greater than 90% TBSA were treated successfully with auto-ESS. ESS were prepared from split-thickness skin biopsies collected after enrollment of the burn patients by Informed Consent into a study protocol approved by the local Institutional Review Board. Subject #130 was a 10 year-old male who sustained 94% TBSA burns, and Subject #131 was a 2 year-old female who sustained 90% TBSA burns. The injuries were all full-thickness, and occurred in separate building fires in 2007. ESS were prepared from autologous keratinocytes and fibroblasts which were isolated from split-thickness skin, cultured, and cryopreserved for later use. Cells were combined with collagen-based sponges, and incubated at the air-liquid interface to promote formation of epidermal barrier. ESS and split-thickness skin autograft (AG) were applied in a matched-pair design with each patient serving as their own control. The first application of ESS was compared to AG for all end points, and subsequent applications of ESS were added to the first and quantify device efficacy. Data collection consisted of photographs, area measurements of donor skin and healed wounds at post operative days (POD) 14 and 28 after grafting, and healed tissue biopsies as available. Data are expressed below as mean values for these two subjects for: A) % area closed at post-operative day (POD) 14, B) %TBSA closed at POD 28, and C) ratio of closed to donor areas at POD 28. Due to the small sample size, mean values were calculated, but no statistical analyses were performed.

Figure 5. Comparative grafting of engineered skin substitutes (ESS) and meshed, split-thickness skin autograft (AG). Left) ESS is applied readily with forceps and adheres rapidly to wounds. Right panel) Comparative grafting of ESS and AG on the anterior torso of subject #130. Scale in centimeters.

Prior to treatment with ESS, wounds were excised, and grafted with either meshed, allograft skin or Integra Dermal Regeneration Template. Two-stage grafting was performed in which the allograft or silicone layer of Integra was removed, and wounds were treated overnight at two-hour intervals with alternating irrigations of 5% Sulfamylon solution and double antibiotic solution (200 U/mL polymyxin B and 40 μg/mL neomycin) 32. The following morning, the dressings were removed in the operating room, hemostasis was obtained with electrocautery and compression. Autograft skin was harvested at a thickness of 0.010-0.012 inches thickness, and meshed and expanded 1:2. ESS were applied with a dressing of N-Terface, and AG was applied directly to the prepared wounds. Grafts were stapled to the wounds, dressed with fine-meshed gauze and bulky gauze with perforated red rubber catheters and secured either with a Spandex stent or with elastic wrap bandages. Sites were irrigated for five days with a formulation of non-cytotoxic antimicrobial agents 1 at a dosage of 1mL/cm2 three times per day. Dressings were changed on POD 2. On POD 5, wet dressings were discontinued, and all dressings and staples were removed. Open areas of ESS were dressed with a topical ointment consisting of equal parts Neosporin, Bactroban and Nystatin on Adaptic. Open areas of AG were dressed with a topical cream consisting of equal parts Silver sulfadiazine, Bacitracin and Nystatin on Adaptic. Keratinized areas of ESS were treated with moisturizing lotion (i.e., Curel) beginning at POD 11, and moisturizing cream (i.e., Eucerin) was applied to AG beginning at POD 7. Both graft types were treated according to the AG protocol beginning at POD 15. Results: Subject #130 received 12 applications of ESS over 4 months, and Subject #131 received 7 applications of ESS over 3 months. Average % engraftment (dry epithelium) at POD 14 was 72.4% for ESS and 96.9% for AG. Partial regrafting was performed in 8 of 12 ESS sites (66%) for Subject #130, and 4 of 7 ESS sites (57%) for Subject #131. The average ratio of closed wound area to donor skin area at POD 28 was 125.5 for ESS, compared to 4.0 for AG. Average %TBSA closed at POD 28 was 51.4% for ESS, and 40.6% for AG. Physical therapy was resumed beginning at POD 7, and ESS which was healed at POD 28 did not blister or ulcerate subsequently. Patients wore pressure garments over all treated areas. Pigmentation of areas treated with ESS was deficient, but pliability of healed skin was acceptable. Figure 6 shows images of Patient A at the time of hospital discharge, 187 days after the first treatment with autologous engineered skin.

Conclusions: These results illustrate that ESS offers the potential to reduce requirements for donor skin harvesting for grafting of excised, full-thickness burns involving most of the TBSA. Survival of these two patients after treatment with ESS is consistent with previous findings that autologous engineered skin is associated with reduced harvesting of donor skin autograft 1, and decreased mortality in matched patient populations 27. Availability of ESS for treatment of extensive, deep burns may reduce time to wound closure, morbidity and mortality in this pediatric patient population.

Figure 6. Subject #130, a 94% TBSA burn with healed ESS and AG at POD 187. Top panels) Torso and arms. Bottom panels) Legs. Wounds close rapidly because of epidermal keratinization in vitro, and do not blister because of basement membrane formation. igmentation of most areas treated with ESS was deficient, but pliability of healed skin was acceptable. The patient wore pressure garments over all treated areas. Application as non-expanded sheets reduces granulation and scar to generate a relatively smooth surface. Scale in cm. 

 

Figure 7. Photos of subject #131 at post-burn day 370 after grafting of engineered skin substitutes (ESS) and meshed, split-thickness skin autograft (AG) for treatment of 90% TBSA burns. Left panels, anterior) ESS provides stable wound closure and generates skin which is smooth, soft and strong. Right panels, posterior) Pliability of healed skin allows free ambulation. Scales in centimeters.

26

 

 

 

Figure 8. Quantitative outcomes of ESS and AG in treatment of two pediatric patients with ≥90% TBSA burns. A) % of treated area closed at POD 14 was 72.4% for ESS and 96.7% for AG; B) % TBSA closed at POD 28 was 51.4% for ESS and 40.6% for AG; and, C) Closed:donor ratio at POD 28 was 125.5 for ESS compared to 4 for AG. Importantly, the closed:donor ratio of ~125 (C) demonstrates a significant reduction in donor skin harvesting for patients treated with ESS, and defines a new medical benefit for these patients.

 

Taken together these studies provide a solid foundation for prospective studies with ESS in adults, and for increased availability through fabrication by a commercial manufacturer. The requested funding is anticipated to expedite the availability of ESS to wounded soldiers, to reduce their suffering during hospitalization, decrease morbidity and to improve their long-term quality of life.

 

4.3 ESS process

 

The technology transfer of the ESS manufacturing process took place in 2006 and included activities such as set-up of all project-specific parts, technician training on cell isolation, cell expansion and inoculation, lab set-up, and acquisition of all necessary equipment. Technical transfer and training also included release assays and in process testing procedures, including histology and surface hydration testing.

 

4.3.1 Current manufacturing process

 

Lonza’s Cell Therapy Process Development staff has made significant improvements to the process since its acquisition. Lonza has qualified new raw material suppliers and improved formulations, including changes to the media, antibiotics, and a decrease in growth factors used in the process. Lonza also made changes to cell culture vessels and purchased a custom-made material for ESS lifting. The current process begins with an isolation of keratinocytes and fibroblasts from a donor biopsy. Each cell type is expanded separately until 75-95% confluent (6 to 8 days). This stage of culture is referred to as Passage 1 (P1). At this point, a portion of the cells are expanded through P2 and P3, with a passage occurring every 6 to 8 days. The remaining portion of the cells is cryopreserved for a second round of expansion and ESS production. Each patient receives two ESS grafts approximately two weeks apart. When the expanded cells reach confluence in the P3 stage, they are harvested and inoculated onto pre-hydrated collagen biopolymers. On the first day of inoculation, fibroblasts are harvested, concentrated, and seeded onto biopolymers in a dropwise fashion at a specified concentration. The fibroblasts absorb onto and migrate through to the lower surface of the biopolymer over the next 24 hours. The next day, keratinocytes are harvested, concentrated, and put onto the same biopolymers in a dropwise fashion. The keratinocytes continue to grow on the upper surface of the ESS. Inoculated grafts sit on sterile lifting frames so that they receive the correct amount of moisture on the lower surface (fibroblast surface) and little to no moisture on the upper surface (keratinocyte inoculated surface). The biopolymer is a collagen-glycosaminoglycan (CAG) substrate produced by lyophilization of the collagen-GAG slurry, followed by dehydrothermal treatment and terminal sterilization by gamma irradiation. It has been and will continue to be produced by Lyophilization Services of New England (a FDA registered cGMP manufacturer with expertise in collagen substrate production), or a comparable supplier. An outline of the process is in Figure 9.

27

 

Figure 9.

Process Flow

 

28

 

4.3.2 Development of Manufacturing Batch Records (MBR) for cGMP manufacture.

 

Beginning in 2006, Lonza developed a comprehensive set of standard operating procedures for manufacturing for ESS. These procedures are referred to as Manufacturing Batch Records on which each procedural step is specified and verified for each manufacturing run. Examples of MBRs include, but are not limited to:

 

4.3.2.1	Skin biopsy collection and shipping
4.3.2.2	Cell isolation and primary culture of fibroblasts and keratinocytes
4.3.2.3	Harvesting of cultured cells for:
		Serial passage
		Cryopreservation in liquid nitrogen
4.3.2.4	Recovery from cryopreservation and subculture
4.3.2.5	Expansion of cell populations
4.3.2.6	Inoculation of cells onto biopolymer sponges
4.3.2.7	Incubation at the air-medium interface
4.3.2.8	Quality Assurance testing by histology and epidermal surface hydration
4.3.2.9	Sterility testing for product release

 

These MBR procedures have been organized into a process flow diagram which is shown on the following page (Figure 9). This process has been used repeatedly in development and engineering runs at Lonza to reproduce the ESS technology, and to upgrade the procedures and reagents used previously to meet standards for current Good Manufacturing Practices. This process and these procedures will be combined with documentation for facilities validation and reagent certifications to submit to FDA for clearance to manufacture ESS for the proposed clinical trial.

 

4.3.3 Development Runs at Lonza

 

The ESS project at Lonza has included research and development for process improvements. Currently Lonza has performed 6 development experiments, 9 full engineering runs, and 22 partial engineering runs. Developmental research at Lonza has been focused on the reduction of animal origin materials, media optimization, improved cell yield from isolation, reduction of growth factors, and comparable or improved growth of keratinocytes and fibroblasts before and after inoculation of ESS. Engineering runs and experiments varied in size and remained small for training purposes. Expected yields can be based on the following results:

 

Development Run Example A Biopsy size= 30 cm2 Number of days in culture: P1= 6 P2= 5 P3= 5 on ESS= 13 Total Number of days before release= 30 Total ESS made from 2E6 fibroblasts and 4E6 keratinocytes= 8 Potential total amount of ESS produced per lot= 2385 cm2 Total skin expansion = ESS area / biopsy area = 2385 / 30 = 80 fold

Development Run Example B Biopsy size= 42 cm2 Number of days in culture: P1= 7 P2= 6 P3= 6 on ESS= 14 Total Number of days before release= 33 Total ESS made from 2E6 fibroblasts and 4E6 keratinocytes= 4 Potential total amount of ESS produced per lot= 4706 cm2 Total skin expansion = ESS area / biopsy area = 4706 / 30 = 157 fold

29

 

Estimated Model for Clinical production lots Biopsy size= 30 cm2 Number of days in culture: P1= 6 P2= 6 P3= 6 on ESS= 13 Total Number of days before release= 31 Total ESS made from 2E6 fibroblasts and 4E6 keratinocytes= 20 Potential total amount of ESS produced per lot= 3600 cm2 Total skin expansion = ESS area / biopsy area = 3600 / 30 = 120 fold

 

 

Figure 10. ESS produced at Lonza Walkersville, Inc.Left, lower) A custom-designed culture dish for incubation of ESS at the air-liquid interface to promote formation of epidermal barrier. This dish will allow a larger format of ESS which will reduce linear scars between grafts, and simplify surgical application. Dish width = 24 cm. Left, upper) Individual ESS device with corners removed for histology. Dish diameter = 15 cm. Right) Multiple devices are exposed to the atmosphere to measure surface hydration. Dish diameter = 15 cm.

4.3.4 Production Plan

 

Lonza plans to carry out additional development runs including final engineering runs. Lonza will then be prepared for the isolation and expansion of a 30-40 cm2 biopsy per patient over a 4-6 week time period into multiple ESS grafts. Two production runs of ESS grafts will result in a final total area of 3,600 to 4,200 cm2 for each patient. The ESS grafts will be applied in two separate procedures approximately two weeks apart. The second cell expansion will be performed using the cryopreserved P1 cells (as referenced in section 4.3.1).

 

4.3.5 Status of GMP process and compliance

 

Lonza has developed this product and manufactured ESS grafts that are acceptable according to histology testing, surface hydration testing, visual inspection and safety testing procedures. Product made at LWI has not yet been used in a clinical trial or transplanted to patients. All MBRs exist in draft form and will be finalized and entered into Lonza’s document control system before being used in clinical production. Other key steps to compliance and certification of the product will include in-house tech transfer of the remaining release tests to the Cell Therapy QC group. Once documentation and training are complete, Lonza production staff will perform 2-3 Engineering Runs. The entire process to initiation of GMP manufacturing will take about 12-16 weeks.

30

 

4.3.6 Quality

 

The LWI Quality Department is composed of Quality Assurance, Regulatory Affairs, Document Control, Label Control, Inspection, Validation, and Quality Control. This department operates independently from management and production. Responsibilities include in-process monitoring and the establishment of standards for personnel, facilities, procedures, equipment, testing, and record keeping. Lonza Walkersville Inc. maintains dedicated Quality Assurance and Quality Control staff to monitor all processes.

 

The Quality Assurance group supports the manufacture and release of product from Cell Therapy. The Quality Control laboratories are divided into Cell Biology, Microbiology and Molecular Biology and staffed by a group of over 65 individuals. All activities are coordinated by a centralized receiving and logging process to assure cGMP compliance. An additional testing lab is available within the designated manufacturing area to provide additional in-process support.

 

4.4 ESS product testing rationale and product tests. The in-process and final release testing for ESS is described below

 

4.4.1 Bioburden

 

Bioburben testing is performed on the biopsy transport medium using a protocol in accordance with USP<61> Microbial Test Limits. Identification of microbial agents detected will be reported as “For Information Only” (FIO).

 

4.4.2 Sterility

 

Grafts are tested for sterility during maturation (6-7 days prior to product release) and at final release. Samples are generated by pooling of 1 ml spent medium from 100% of the grafts followed by testing in accordance with USP<71> Sterility using the membrane filtration method. An additional 1 ml sample is retained for retesting if necessary.

 

4.4.3 Surface Electrical Capacitance (SEC)

 

SEC is measured with a Nova Dermal Phase Meter (DPM 9003) as a quantitative surrogate index of epidermal barrier formation in the ESS. Four locations are tested on every graft on days 6 or 7 and a second test on days 8 or 9 of incubation. The data are averaged and demonstration of a time-dependent SEC decrease yields a passing graft.

 

4.4.4 Histology

 

Evidence of a stratified epidermal substitute attached to a dermal substitute populated with fibroblasts indicates ESS acceptable for grafting. To minimize graft handling, 10% of grafts will be tested in a non-destructive manner (as can be seen in Figure 10).

 

4.5 ESS Summary

 

The product is an engineered skin substitute (ESS) consisting of autologous skin cells within a degradable biopolymer substrate. Isolated epidermal keratinocytes and dermal fibroblasts are cultured in nutrient media to promote population expansion. The cells are separately seeded onto a biopolymer substrate fabricated from collagen and carbohydrate polymers (glycosaminoglycan). The ESS device has been shown in preclinical studies to generate a functional skin barrier and in clinical studies to promote closure and healing of burns. This is the only medical device known at present for the treatment of full-thickness burns with autologous cells combined with a polymeric substrate.

 

With extensive cell therapy expertise and manufacturing capabilities, Lonza is well positioned to produce ESS at the Walkersville, MD facility. Based on yields generated during engineering runs and development runs, it is projected that cells isolated from a 30-40 cm2 biopsy, would generate an approximate yield of 3600 cm2 per lot of ESS material. Production of ESS would be staggered using two lots to produce enough product per patient in 6-8 weeks.

31

 

5. Proposed Research and Methods:

 

5.1 Specific Aims. The main objective of the work that will be supported by this supplemental funding is to expedite availability of ESS by performance of a limited study (8-10 subjects) under an Investigational Device Exemption (IDE) protocol, with ESS generated by the commercial manufacturer, Lonza Walkersville, Inc (LWI). ESS remains an investigative device which has been studied previously as described above. Toward these objectives, two specific aims are proposed:

 

5.1.1 Planning for delivery of ESS (trade named, PermaDerm) to a qualified burn center from Lonza Walkersville, Inc. for application to burn patients under an IDE protocol. Availability requires: a) verification of device manufacture under standards for current Good Manufacturing Practices (cGMP); b) demonstration of quality assurance criteria for product release; c) development of packaging materials and shipping protocols; d) development, validation and FDA acceptance of a clinical trial protocol; e) compliance systems through the AFIRM Clinical Trials Office at Case Western Reserve University; and, f) permission from FDA to proceed with the clinical study.

 

5.1.2 Comparison of ESS and meshed, split-thickness autograft in a controlled, prospective study. The proposed study will follow an open-label, randomized, matched-pair, internally-controlled design similar to previous studies, but in adult burn patients. The hypothesis of the study is that ESS provides a quantitative reduction in donor skin required to complete wound closure, and scar which is not statistically different from, or better than meshed and expanded split-thickness skin autograft. End points for the study are described below in section 6.

 

5.2 Research Plan.

 

5.2.1 Diagram of the research plan.

 

 

 

Figure 11. Diagram of the research plan. Lonza Walkersville, Inc. (LWI) is the applicant and study sponsor. The study design (top) will be submitted to the AFIRM Clinical Trials Core (“CTC”; Dr. Gerson) for review, and addition of compliance procedures (e.g., DSMP, DSMB). The AFIRM-CTC will advise a clinical research organization (CRO) which will assemble the Sponsor’s and Investigator’s Brochures, Case Report Forms, and the study notebook for each subject. LWI will submit the IDE protocol to FDA. After FDA acceptance, the CRO will work directly with the clinical performance sites to submit protocols to the local Institutional Review Boards, and then to the Human Subjects Research Review Board (HSRRB) at USAMRMC at Ft. Detrick. After all permissions are obtained, the CRO will serve as the sponsor’s agent to monitor enrollment and treatment of subjects, data safety and study reporting. Manufacturing protocols (bottom) will follow established procedures at Lonza for regulatory acceptance.

32

 

5.2.2 Device manufacture under cGMP processes. LWI will confirm completion of Standard Operating Procedures (SOPs) for manufacture of ESS under conditions which comply with standards for current Good Manufacturing Practices (cGMP). The batch size (e.g., 2,000 cm2) will determine the specific cGMP suite which LWI will assign for this project. After assignment, the suite will be equipped, staffed, and validated for manufacturing. This step in the project is expected to require about 3 months.

 

5.2.3 Validation of quality assurance (QA) criteria for product release. Quality assurance criteria for product release will include both general safety criteria for medical devices (sterility, mycoplasma, endotoxin), and specific criteria for product potency (epidermal barrier, cellular organization, device thickness) which constitute criteria for product release. The general criteria are routine practices at LWI, and will be included in the facility activation. The specific criteria have been included in engineering runs at LWI, and SOPs for those assessments have been developed.

 

5.2.4 Validation of clinical study design through the RCCC-AFIRM Clinical Trials Office. The study design will be adapted from previous studies with engineered skin substitutes (see section 6). That design will be submitted for review to the RCCC-AFIRM Clinical Trials Core under the direction of Stanton Gerson, MD in the Center for Stem Cell and Regenerative Medicine at the Case Western Reserve University. The AFIRM-CTC will format the documents, review the safety and efficacy end points of the study, and consider any revisions which may be needed to the statistical analyses of the data. The AFIRM-CTC will assure compliance of the study with standards for current Good Clinical Practices (cGCPs).

 

5.2.5 Engagement of a Clinical Research Organization (CRO) for daily management of the clinical trial. A CRO with experience in medical device evaluation will be selected and retained to serve as the sponsor’s agent and manager of the clinical trial. The CRO will have responsibilities to assemble and finalize the study documents, including but not limited to: a) clinical trial protocol; b) the Investigator’s Brochure; c) clinical monitoring plan; and, d) approvals from the Institutional Review Boards (IRBs) of performance sites, and the Human Subjects Research Review Board of the DoD.

 

5.2.6 Regulatory protocol development and submission. LWI will be responsible for device manufacturing, process validation, regulatory submissions, and clinical performance at one or more qualified burn center(s). Lonza has initiated technology transfer for manufacture of the ESS device (trade named PermaDerm™) under cGMP (i.e., clean room) conditions. Devices produced by Lonza have not yet been compared directly to devices fabricated in the Dr. Boyce’s research laboratories in Cincinnati, OH. Comparability testing will be performed as required by FDA with support from Lonza as a contribution in kind to the project.

 

5.2.7 Commitment of sites for clinical performance of the study. Because the AFIRM program is directed toward new therapies for wounded warfighter, the first choice for a clinical study site for burn repair is the Burn Unit of the US Army Institute for Surgical Research (ISR) at the Brooke Army Medical Center of Fort Sam Houston in San Antonio, Texas. In conference with Steven Wolf, MD, director of clinical investigations at the ISR, some, but perhaps not all, of 8-10 subjects for this proposed study may be available for accrual within the planned study enrollment period of one year. Drs. Wolf and Blackbourne have committed for the ISR to participate in this study (see appendix). In the event that a sufficient number of subjects is not expected to be available at the Burn Unit of the ISR, one additional site will be recruited for subject enrollment. Several burn centers (Indianapolis, Seattle, Los Angeles, Phoenix, Baltimore, Washington, DC) have sufficient census records to meet the requirements of the study, and will be interviewed as candidates for participation. Dr. Boyce will assist LWI with this recruitment.

 

5.2.8 Timetable for availability of autologous ESS for massive burns.

 

As described above, the requested support will be applied to activation of cGMP manufacturing of ESS from Lonza Walkersville, Inc., and to planning for clinical availability to the USAISR, or other qualified burn center. Below is a Gantt chart (Table 2) which summarizes the schedule for completion of regulatory and operational requirements for the project, based on the assumption that all parties make reasonable efforts to complete the project according to this timeline. Assuming start of funding by 1 Jul 2009, and with the collaborative efforts of all parties, the applicants believe this goal can be reached in the second calendar quarter of 2010.

33

 

Table 2. Schedule for project performance.

 

 

 

6. Clinical Trial:

 

Planning of the clinical study trial design will be based on previous studies under IDE G980023, with revisions according to advice of the investigators at the USAISR, the RCCC-AFIRM Clinical Trials Core Facility, and discussions with FDA.

 

6.1 Experimental design and end points. A matched-pair comparison format will be used to evaluate ESS and conventional split-thickness skin graft for closure of full-thickness, excised burns. This study will be performed in a prospective, randomized fashion with each pair in the same patient on wounds of similar size and depth. For each administration of ESS to a wound site, a comparative site will be treated with meshed split-thickness autograft skin (AG). Application of the specific treatment to site A or B will be randomized prior to initiation of the study. Up to 10 randomized pairs will be generated.

 

Wound treatment and dressings. Burns will be debrided by excision as early as possible after burn injury and wounds will be covered with fresh or cryopreserved cadaver allograft or Integra Dermal Regeneration Template™ (IDRT). One day prior to surgery, allograft or the silicone cover on IDRT will be removed, the wounds will be irrigated overnight with an appropriate antimicrobial solution (i.e., 5% wt/vol Sulfamylon solution), and the graftswill be applied the following day32Wound preparation for each site will be recorded at the time of surgery. Wound dressings will be administered as described previously26,33,34, using a non-adhesive porous dressing (ie., N-Terface, Winfield Laboratories) in direct contact with the cultured skin covered with fine mesh gauze and bulky dressings. Dressings for skin substitutes will be irrigated32 with nutrients, plus non-cytotoxic antimicrobial drugs (i.e., GU irrigant, polymyxin B, mupirocin, ciprofloxacin, amphotericin B)35 over the N-Terface for 5 days. On POD 5, wet dressings will be discontinued, and changed into an ointment-impregnated synthetic mesh (i.e., Adaptic) until complete wound closure is achieved. For ESS a topical ointment consisting of equal parts Neosporin, Bactroban and Nystatin (NBN) will be used.

 

6.2 Enrollment criteria.

 

6.2.1 Medical indication. Patient has full-thickness burns equal to, or greater than, 50% of the Total Body Surface Area.

 

6.2.2 Medical conditions. Patient is not septic, and is expected to require ongoing skin grafting for a period longer than 3 weeks after collection of a skin biopsy to generate ESS

6.2.3 Age and gender. Age 18 years or older, either gender.

 

6.2.4 Social requirements. Patient is not pregnant or lactating, not a prisoner, and not mentally incompetent.

 

6.2.5 Completion of Informed Consent Form, and Health Insurance Portability and Accountability Act (HIPAA) forms.

34

 

6.3 Study end points for safety. Device safety will be assessed in comparison to AG by: a) frequency of regrafting on or before POD 28; b) anticipated and unanticipated adverse device effects; and c) histopathologic interpretation of healed wound biopsies. Late graft stability will be recorded but is not a safety endpoint.

 

6.3.1 Frequency of regrafting of comparative sites will be recorded at POD 28. Each site will be scored for regrafting as None, Partial, or Total. Comparative sites will be randomized according to a pre-determined schedule prior to the placement of the ESS.

 

6.3.2 Anticipated and unanticipated adverse device effects will be adapted from a list of approved by FDA from the previous study of the prototype device (see section 6.6). Those events will be incorporated into the Investigators Brochure, and recorded in the Case Report Forms. Serious adverse events will be reported within 72 hrs to the sponsor, local IRB, AFIRM Clinical Trials Unit and FDA IDE office.

 

6.3.3 Histopathologic interpretation will be made by a dermato-pathologist from histological slides of biopsies (up to 4/pt during first year after grafting) taken from healed wounds. Interpretations of the epidermis, the dermal-epidermal junction, and dermis will be made according to dermato-pathological standards.

 

6.4 Study end points for efficacy. The primary end point for device efficacy is a statistically significant increase in the donor skin area of the closed wound at post operative day (POD) 28 divided by the area of the donor skin used to close that area (section 6.4.1). Secondary end points for efficacy include: a) Percentage engraftment (i.e., closed wound) at POD 14 (section 6.3.2); b) Percentage TBSA closed at POD 28; and c) scoring of scar qualities of erythema, pigmentation, pliability and scar height by the Vancouver Scar Scale.

 

6.4.1 Donor skin expansion ratio. This ratio of healed wound area to biopsy skin area allows determination of donor site utilization. For ESS donor skin area will be traced at the time of isolation of cells to initiate device fabrication. For AG, donor skin area will be traced after application to the comparative site, and the mesh ratio (i.e., 1:2; 1:3; 1:4) will be recorded. The areas of the tracings will be determined by computer assisted image analysis. The donor skin expansion ratio will be calculated for each treatment site from at POD 28 as:

 

Closed wound area / donor skin area = donor skin expansion ratio

 

The donor skin expansion ratio for ESS, the prototype of ESS has been reported to be more than 10X greater than for AG 27,28. This outcome defines a primary medical benefit of ESS compared to AG in the treatment of extensive, full-thickness burns.

 

6.4.2 Percentage engraftment is determined by tracings of the grafted sites at POD 14 followed by planimetry. The tracing at POD 14 allows determination of % engraftment as healed area/treated area X 100.

 

6.4.3 Percentage TBSA closed is determined by dividing the absolute values of all of the areas closed, as measured by tracings and planimetry at POD 28, by the absolute value of the TBSA, and multiplying X 100. The percentage TBSA covered with split-thickness skin autograft is determined by subtracting the absolute value of the area closed with ESS from the absolute value of the area grafted, dividing by the absolute value of the TBSA, and multiplying X 100.

 

6.4.4 Scar qualities by the Vancouver Scar Scale 36. Both sites will be scored for erythema, pigmentation, pliability and scar height on semi-quantitative, ordinal scales. Erythema ranges from 0 (normal) to 3 (purple), pigmentation is rated from 0 (hypo) to 3 (hyper), scar height is scored from 0 (flat) to 3 (>5 mm), and pliability from 0 (normal) to 5 (contracture).

 

6.4.5 Long term efficacy observations. This trial is designed as a short term feasibility study. However, recipients will be monitored at 12 months after application for efficacy parameters including overall medical condition and complications referable to the burn; skin coverage assessed by the Vancouver Scar Scale, frequency of regrafting and evidence of inflammation by histopathology.

35

 

6.4.6 Table of safety and efficacy end points for assessment of ESS

 

Table 3. Data collection schedule for assessment of ESS and AG in burn patients.

 

Primary Analyses at 4 weeks & 1 year after comparative treatment
Type	Endpoint	POD	-7	0	7±3	14±3	28±3	365±30
Safety	Microbiology cultures of wound		X	X	X	X
Efficacy	% Engraftment					X
Efficacy	Donor skin expansion ratio						X
Efficacy	% TBSA closed						X
Efficacy	Vancouver Scar Scale						X	X
Efficacy	Photography			X	X	X	X	X
Safety	Frequency of regrafting						X
Safety	Histopathology						X	X

 

6.5 Statistical power analysis.

 

Based on the data collected for determination of device efficacy as the ratio of closed wound areas to donor skin areas, a statistical power analysis was performed using data from 54 subjects previously reported 2. The mean ratio of the autologous ESS device was 63.9. This value for ESS was compared to a single value of 4.0 which was assigned to split-thickness skin autograft (AG) because the combined areas treated with AG were not measured directly, but were calculated by subtraction of the total area closed with ESS from the total burn area. In practice, the ratio for AG was actually closer to 2.0. Table 3 below shows that for power values of 0.95, 0.90, 0.80, and 0.50, the sample sizes required are 11, 10, 8 and 5 respectively. Therefore, it is expected that efficacy of the ESS device can be demonstrated in less than one year of enrollment of adult subjects at the USAISR, and at another qualified burn center which treats adults.

 

Table 4. Statistical power analysis for clinical trial of ESS compared to meshed autograft skin based on expansion ratios. This analysis was performed using a 2-sided, one sample paired T-test.

 

Power analysis for Prospective Trial of ESS
Alpha	Beta	power	ESS mean	AG mean	difference	Std	n
0.05	0.05	0.95	63.93	4.0	59.96	48.8	11
0.05	0.10	0.90	63.93	4.0	59.96	48.8	10
0.05	0.20	0.80	63.93	4.0	59.96	48.8	8
0.05	0.50	0.50	63.93	4.0	59.96	48.8	5

 

This analysis makes two important assumptions that the ESS device performs comparably:

a) to the ESS device used in previous studies; and, b) in adults as in pediatric subjects.

 

Based on this analysis and the resources available, this initial trial proposes to treat no less than 8, and no more than 10 subjects as a proof-of-principle (i.e., pre-pivotal) study. Lonza will deliver up to 40,000 cm2 (4.0 m2) of autologous engineered skin for the proposed study.

36

 

6.6 Process for review, performance and compliance of FDA-regulated, clinical trials through the

AFIRM consortium.

 

Several considerations for study validity and regulatory compliance are required prior to initiation of clinical trials. This study with engineered skin is one example of a general process for clinical trial and technology transfer. The RCCC-AFIRM consortium has established a Clinical Trials Core located at Case Western Reserve University and the Cleveland Clinic Foundation for data management and regulatory oversight of studies it supports.

 

6.6.1 Data Safety Monitoring

 

Each clinical trial site will have its own management plans for data safety monitoring. A CRO will provide external oversight of data collection and audit compliance with all aspects of the study. If two sites are used for the clinical trial, an independent Data Safety Monitoring Board will be established to review adverse event (AE) reporting during the conduct of the trial. This independent body will report information to the PI, the AFIRM Clinical Trials Core, and will advise on reporting to the institutional IRBs and the FDA.

 

The AFIRM Clinical Trials Core will coordinate the multi-site clinical data using a web-based CFR Part 11-compliant electronic database for recording and verification of data collection in real time. The Core will monitor IRB and FDA approvals, amendments, and annual reports. Updated consents and clinical protocol will be available for online access. It will maintain records for subject registration, patient demographics, accrual dates, key event dates, including graft placement and timed follow-up assessment results. It will record all clinical data on electronic Case Report Forms (CRFs).

 

Major outcome and study endpoint parameters will be monitored, including overall medical condition and complications referable to the burn, such as skin coverage assessed by the Vancouver Scar Scale, frequency of re-grafting, and evidence of inflammation by histopathology. Adverse and serious adverse event data collected at the study sites will be maintained and monitored. Serious adverse event definitions will be compliant with national reporting standards and will use conventional definitions for burn patients. All organ assessment and adverse events will be recorded during the 28 d observation period after graft placement.

 

The Clinical Trials Core database will be available under secure access for audit, and will be reported to the AFIRM executive committee, individual PIs and the device supplier, Lonza Walkersville, Inc.

 

6.6.2 Data Safety Monitoring Plan

 

Each institution will utilize its own Data Safety Monitoring Plan. The following list outlines general components that will be included:

 

• Review by a Data, Safety and Toxicology Committee (DSTC)

• Review of annual and special reports

• Management of Oversight Conflict of Interest

• Plans for Assuring Compliance with the Requirements Regarding Reporting of Adverse Events

• Plans for Assuring Temporary or Permanent Suspension of Clinical Trial Protocols

• Plans for Assuring Data Accuracy and Protocol Compliance

• Compliance Audits

• Quality Assurance (Database Edit Checking, Chart Audits, Protocol Compliance, Registration On-Study, Internal Audit Process)

• Management of Multi-center Trials (Industry and Academic trials)

• Study Coordinator Reviews

• Corrective Actions

37

 

6.6.3 Reviews required prior to clinical protocol approval and activation:

 

• AFIRM scientific review committee.

• USAMRMC clinical trials office at Fort Detrick.

• Review and IRB approval by performance site (ISR and other).

• Review and permission to proceed from FDA.

• The pre-IDE meeting with the FDA will be scheduled within three months of project activation.

 Documents submitted will be used to develop specific questions for the pre-IDE meeting. At that point, LWI will submit to FDA the IDE with final protocol, final manufacturing SOPs for the PermaDerm product and monitoring plans for the trial. We expect to have provisional IRB review pending IDE review by the FDA. Once the IDE is allowed, after the 30 day review period, we will seek final site approval of the IRB.

• Data collection, internal review and reporting to FDA. Complete enrollment and file final report.

 

The application of resources from this supplement is anticipated to expedite the availability of ESS for treatment of bured soldiers in 12-18 months from initiation of funding, rather than 24-36 months if supplemental support is not available. The expedition of delivery of ESS is expected to demonstrate proof-of-principle for life-saving medical benefits and improvements in outcome for military and civilian patients with massive burn injuries.

38

 

7. Abbreviations and Acronyms:

 

AFIRM:	Armed Forces Institutes for Regenerative Medicine
AG:	split-thickness skin AutoGraft
CBSW:	Cambrex BioScience Walkersville, Inc.
cGCP:	current Good Clinical Practices
cGMP:	current Good Manufacturing Practices
CRFs:	Case Report Forms for the study
CTC:	Clinical Trials Core facility of the RCCC-AFIRM at Case Western Reserve University
ESS:	autologous Engineered Skin Substitutes
FDA:	United States Food and Drug Administration
HSRRB:	Human Subjects Research Review Board
HUD:	Humanitarian Use Device
IDE:	Investigative Device Exemption
IDRT:	Integra Dermal Regeneration Template™
IRB:	Institutional Review Board
LWI:	Lonza Walkersville, Inc.
ORCRA:	Office of Research Compliance and Regulatory Affairs
ORP:	Office of Research Protections
PD:	PermaDerm™; trade name for engineered skin substitutes
PMA:	Pre-Market Approval
POD:	Post-operative day
SEM:	Standard error of the mean
TBSA:	Total Body Surface Area
USAISR:	United States Army Institute for Surgical Research
USAMRMC:	United States Army Medical Research and Materiel Command
DMEM:	Dulbecco's Modified Eagle Medium
KGM-CD:	Keratinocyte Growth Medium - Chemically Defined
UCMC:	ESS Keratinocyte Growth and Culture Mediu

39

 

8. References

 

1. Boyce ST, Kagan RJ, Greenhalgh DG et al. Cultured skin substitutes reduce requirements for

harvesting of skin autograft for closure of excised, full-thickness burns. J Trauma 2006; 60:821-829.

2. Boyce ST, Greenhalgh DG, Palmieri T et al. Autologous cultured skin substitutes reduce requirement for split-thickness skin autograft in treatment of excised, full-thickness burns. J Burn Care Res 2006;27:S59.

3. Boyce ST, Simpson PS, Kagan RJ. Survival of burns involving 90% of the total body surface area after treatment with autologous engineered skin substitutes. Proc of 2008 Army Science Conference . 2008.

4. Hall JR. The total cost of fire in the United States through 1991. In: Quincy MA ed. National Fire Protection Association. 1993:1-20.

5. Shires GT. Evolution of trauma and trauma research. J Trauma 1990; 30:107s-115s.

6. American Burn Association. National Burn Repository: 2005 Report. 2006. American Burn

Association.

7. The Burn Foundation. Burn incidence and treatment in the United States. www.burnfoundation.org. 1999. Philadephia, PA.

8. Renz E, Barnes S. Long-range air transport of combat-related burn casualties. Proc ATACCC . 2007.

9. Hansbrough JF, Dominic W, Gadd M et al. Burns: critical decisions. Prob Crit Care 1987; 1(14):558-610.

10. Burn Registry Committee ABA. Comparison of hospital days, ICU days, and days on ventilator support. National Burn Repository 2002; 2002:7-35.

11. Kagan RJ. Restoring nitrogen balance after burn injury. Compr Ther 1991; 17(3):60-67.

12. Kagan RJ, Warden GD. Management of the burn wound. Clin Dermatol 1994; 12:47-56.

13. Boyce ST, Supp AP, Swope VB et al. Vitamin C regulates keratinocyte viability, epidermal barrier, and basement membrane in vitro, and reduces wound contraction after grafting of cultured skin substitutes. J Invest Dermatol 2002; 118:565-572.

14. Supp DM, Karpinski AC, Boyce ST. Vascular endothelial growth factor overexpression increases vascularization by murine but not human endothelial cells in cultured skin substitutes grafted to athymic mice. J Burn Care Rehabil 2004; 25:337-345.

15. Goretsky MJ, Harriger MD, Supp AP et al. Expression of interleukin 1α, interleukin 6, and basic fibroblast growth factor by cultured skin substitutes before and after grafting to full-thickness wounds in athymic mice. J Trauma 1996; 40:894-900.

16. LePoole IC, Boyce ST. Keratinocytes suppress transforming growth factor beta-1 expression by fibroblasts in cultured skin substitutes. Br J Dermatol 1999; 140:409-416.

17. Boyce ST. Epidermis as a secretory tissue. J Invest Dermatol 1994; (editorial)102:8-10.

18. Boyce ST. Method and apparatus for preparing composite skin replacements. University of California San Diego. [5,273,900]. 1993. United States of America.

19. Boyce ST. Apparatus for preparing composite skin replacements. University of California San Diego. [5,711,172]. 1998. United States of America.

20. Boyce ST. Method and apparatus for preparing composite skin replacements. University of California San Diego. [5,976,878]. 1999. United States of America.

21. Boyce ST. Apparatus for forming a biocompatible matrix. University of Cincinnati and Shriners Hospitals for Children. [6,905,105]. 2005. United States of America.

22. Boyce ST. Apparatus for preparing a biocompatible matrix. University of Cincinnati and Shriners Hospitals for Children. [7,452,720B2]. 2008. United States of America.

23. Boyce ST. Method and apparatus for preparing composite skin replacements. University of California San Diego. [363,400]. 1993. Europe.

24. Boyce ST. A surgical device for replacement of skin. University of Cincinnati and Shriners Hospitals for Children. 47359. 2007. European Union.

25. Lonza Group Ltd. Lonza full-year 2008 results. 2009. Lonza Corporation.

26. Boyce ST, Kagan RJ, Yakuboff KP et al. Cultured skin substitutes reduce donor skin harvesting for closure of excised, full-thickness burns. Ann Surg 2002; 235:269-279.

27. Armour AD, Dorr BA, Kagan RJ, Boyce ST. Mortality of acute burns treated with cultured skin substitutes. J Burn Care Res 28[2], S96. 2007.

40

 

28. Supp DM, Wilson-Landy K, Boyce ST. Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice. FASEB J 2002; 16:797-804.

29. Boyce ST, Foreman TJ, English KB et al. Skin wound closure in athymic mice with cultured human cells, biopolymers, and growth factors. Surgery 1991; 110:866-876.

30. Supp DM, Supp AP, Morgan JR et al. Genetic modification of cultured skin substitutes by transduction of human keratinocytes and fibroblasts with Platelet Derived Growth Factor-A. Wound Repair Regen 2000; 8:26-35.

31. Supp DM, Boyce ST. Overexpression of vascular endothelial growth factor accelerates early

vascularization and improves healing of genetically modified cultured skin substitutes. J Burn Care Rehabil 2002; 23:10-20.

32. Warden GD, Saffle JR, Kravitz M. A two-stage technique for excision and grafting of burn wounds. J Trauma 1982; 22:98-103.

33. Boyce ST, Greenhalgh DG, Kagan RJ et al. Skin anatomy and antigen expression after burn wound closure with composite grafts of cultured skin cells and biopolymers. Plast Reconstr Surg 1993; 91:632-641.

34. Boyce ST, Goretsky MJ, Greenhalgh DG et al. Comparative assessment of cultured skin substitutes and native skin autograft for treatment of full-thickness burns. Ann Surg 1995; 222(6):743-752.

35. Boyce ST, Warden GD, Holder IA. Non-cytotoxic combinations of topical antimicrobial agents for use with cultured skin. Antimicrob Agents Chemother 1995; 39(6):1324-1328.

36. Sullivan T, Smith H, Kermode J et al. Rating the burn scar. J Burn Care Rehabil 1990; 11(3):256-260.

 

41

 

10 Facilities and equipment descriptions

 

10.1 General Description

 

Manufacturing of ESS will be performed at Lonza’s facility in Walkersville, Maryland. Lonza is a leading manufacturer of specialty cellular biotherapeutics. Regulatory licenses include:

 

Device Registration Number 1114298

Tissue Registration Number 1114298

CBER License Number 1701

SO Certification Number FM67851

Federal ID Number 953917176

 

Lonza also manufactures endotoxin detection products at the Walkersville facility that are licensed by the FDA. The facility has been regularly inspected in conjunction with licensure of these products. As ESS is not a commercial product, inspection by the FDA has not yet been triggered. are licensed by the FDA. The facility has been regularly inspected in conjunction with licensure of these products. As ESS is not a commercial product, inspection by the FDA has not yet been triggered.

 

10.2 Facilities at Lonza Walkersville, Inc.

 

The Walkersville facility currently has eight completed suites, each ranging in size from 340 to over 800 square feet (+/- 5% ) of core lab space and full suite areas (including staging areas and gown in/ gown out) ranging from 600 to 1380 (see schematic floor plans). Lonza also has additional commercial-scale suites under construction in Walkersville. There are also four suites dedicated to cell therapy manufacturing at the Verviers, Belgium facility. The Walkersville facility currently has eight completed suites, each ranging in size from 340 to over 800 square feet (+/- 5% ) of core lab space and full suite areas (including staging areas and gown in/ gown out) ranging from 600 to 1380 (see schematic floor plans). Lonza also has additional commercial-scale suites under construction in Walkersville. There are also four suites dedicated to cell therapy manufacturing at the Verviers, Belgium facility.

 

ESS production will likely be performed in Cell Therapy Suite A. Room 2306 is a Class 10,000 (ISO Class 7) production suite. It includes four Class 100 biological safety cabinets, 18 humidified incubators, 1 freezer, and 1 refrigerator. Cell Therapy Suite A consists of 6 rooms. It includes 2 class 100,000 airlocks (Room 2302 and 2304) and class 10,000 ingress/egress areas (Rooms 2305 and 2307), a staging area (Room 2317) and a Cell Culture Production area (Room 2306). A materials pass through is built in the wall separating Rooms 2306 and 2305. The walls are composed of fiberglass (FRP board). The floors are made of a troweled epoxy covering. The class 10,000 area is at a positive differential pressure to the outside. The room also includes HEPA filters on the ceiling and return grates on the wall. The utility systems are independent of clean rooms. ESS production will likely be performed in Cell Therapy Suite A. Room 2306 is a Class 10,000 (ISO Class 7) production suite. It includes four Class 100 biological safety cabinets, 18 humidified incubators, 1 freezer, and 1 refrigerator. Cell Therapy Suite A consists of 6 rooms. It includes 2 class 100,000 airlocks (Room 2302 and 2304) and class 10,000 ingress/egress areas (Rooms 2305 and 2307), a staging area (Room 2317) and a Cell Culture Production area (Room 2306). A materials pass through is built in the wall separating Rooms 2306 and 2305. The walls are composed of fiberglass (FRP board). The floors are made of a troweled epoxy covering. The class 10,000 area is at a positive differential pressure to the outside. The room also includes HEPA filters on the ceiling and return grates on the wall. The utility systems are independent of clean rooms.

 

 

 

Figure 12. Typical cell therapy manufacturing suite at Lonza Walkersville, Inc. LWI has several manufacturing suites for cell therapy including all support facilities, a proven track record of compliance with cGMP/ISO standards, and highly trained staff with all of the expertise needed for this project.

42

 

Figure 13. Cell therapy suites at Lonza Walkersville, Inc. The most recent addition to the Walkersville facility includes these 4 suites including 3 class 10,000 suites and a single class B suite. There is more than 2,800 square feet of classified space in this addition

 

10.3 Equipment:

Each of the Cell Therapy facilities is equipped with state-of-the-art manufacturing and analysis equipment to meet the needs of our many clients. This equipment includes everything required for cell culture and expansion, process development, quality assurance, labeling, packaging, and storage and shipping. Equipment is maintained in a validated/calibrated state and remotely alarmed providing 24 hour monitoring. Below is a list of equipment that will be used for the performance of this proposal.

 

Table 5. Manufacturing Equipment:

 

Item	Manufacturer	Qty	Comments
Biosafety Cabinets	Various	20	Between 1 and 4 BSCs are available within each suite
Incubators	Thermo 3950 Thermo 3110 Sheldon Belco	11 51 2 29	These incubators have various capacities and are organized among the Walkersville suites.
Centrifuges	Sorvall	8	Each suite is equipped with a refrigerated centrifuge
Inverted microscope and camera	Various	Several	Each suite is equipped with an inverted or standard microscope.  Cameras can be attached to camera ports for documentation
Refrigerator	Various	Several	Each suite has a minimum of one refrigerator for materials and/or product storage.  Refrigerators are alarmed.
Freezer	Various	Several	Each suite has a minimum of one freezer for materials and/or product storage.  Freezers are alarmed.
Liquid Nitrogen Dewers	Various	Several	Liquid nitrogen is available as needed and is handled through Dewar containers.
Transport Dewers	Various	Several	Carts, dewars and appropriate transport equipment is available to all suites
Cell counter (nucleated)	Guava PCA	Several	Shared resource and QA lab is equipped with cell counters to support suite activities
Controlled rate freezers	Thermo Cryomed	Several	Most suites are equipped with controlled rate freezers

43

 

10.4 Regulatory and Commercialization Plan

 

Since its inception in January 2002, Lonza’s Cell Therapy business unit has developed a global leadership position in cell therapy manufacturing and process development. With the purchase of the ESS device/ESS technology in 2006 and the expansion of manufacturing facilities in Walkersville, Maryland, Lonza is positioned to meet the regulatory and manufacturing requisites for the commercialization of the ESS technology. It is anticipated that the successful completion of the clinical trial design, as described in this proposal, with a limited number of patients, will accelerate commercial launch by providing statistically significant data on the primary efficacy of the ESS technology (i.e., closure rate) in severely burned soldiers within a short timeframe.

 

With the support of the Rutgers/Cleveland Clinic Consortium of the AFIRM network, Lonza will be positioned to commercialize the ESS technology for critically burned patients, for both soldiers and civilians alike. In conjunction with AFIRM consultants, Lonza anticipates scheduling a pre-IDE meeting with the FDA within three months with the goal of an IDE submission by Q2 2009, and enrolling patients in Q1 2010. Lonza is responsible for the IDE submission, and Lonza will make reasonable efforts to prepare the components necessary for the IDE submission, including but not limited to generation of SOPs, preparation of facilities, regulatory compliance of manufacturing, release and shipping, training, preparation for inspections, and pilot or engineering runs as directed by the FDA. Lonza expects to manage and engage a CRO for assistance in two critical areas:

 

1) Author full clinical protocol

2) Trial oversight (e.g. – coordination, implementation, investigator notebooks, case report forms, perform monitoring, reporting, data safety monitoring plan)

 

Additionally, Lonza will continue to work with AFIRM resources to ensure clinical trial compliance. Following the IDE submission and clinical data evaluation, Lonza intends to follow the FDA recommendations on a PMA submission for market approval of the ESS device.

 

The ESS technology will require a manufacturer with significant experience in tissue engineering and the resources to scale to meet commercial needs. Lonza has the commitment and expertise to support these requirements. Since the catastrophic burn market is relatively small (estimated at 2,000 patients annually in the U.S), patients are treated at a small number of centers in the US, requiring a limited investment in sales and marketing efforts. Lonza has a focused technical staff to successfully complete the coordination and training necessary to meet market requirements.

 

Depending on clinical trial outcomes, Lonza is prepared to invest as necessary, to support pursuit of ESS development along the commercial pathway. To ensure that the timeline for marketing approval is as expeditious as possible, Lonza may collaborate with a commercial partner who has specific expertise in the wound care market. Lonza is committed to manufacturing of the ESS device to support patients’ needs.

44

 

11. Patents relevant to autologous engineered skin substitutes.

11.1 Boyce ST. 1993. European Patent 363,400, "Method and apparatus for preparing composite skin replacement". Assignee: University of California.

11.2 Boyce ST. 1993. US Patent 5,273,900, "Method and apparatus for preparing composite skin replacement." Assignee: University of California.

11.3 Boyce ST. 1998. US Patent 5,711,172, “Apparatus for preparing composite skin replacement”. Assignee: University of California.

11.4 Boyce ST. 1999. US Patent 5,976,878, “Method and apparatus for preparing composite skin replacement”. Assignee: University of California.

11.5 Boyce ST. 2005. US Patent 6,905,105, “Apparatus for fabricating a biocompatible matrix”. Assignees:University of Cincinnati and Shriners Hospitals for Children.

11.6 Boyce ST. 2007. European Patent application #47359, "A surgical device for replacement of skin". Assignees: University of Cincinnati and Shriners Hospitals for Children. Claims allowed, 11 Jul 2007.

11.7 Boyce ST. 2008. US patent 7,452,720B2, “Apparatus for preparing a biocompatible matrix”. Assignees: University of Cincinnati and Shriners Hospitals for Children. Claims allowed, July 2008.

11.8 Boyce ST. 2002. US patent application, publication #US2003/0170892, “A surgical device for replacement of skin”. Assignees: University of Cincinnati and Shriners Hospitals for Children. Pending review.

 

12. Intellectual and material property plan. (see sections 1.4 and 10)

45

 

Budget Justification:

 

Personnel – Faculty

The management of this project will be a coordinated effort by three Principal Investigators, one each from:

1. Lonza Walkersville, Inc (LWI) for overall project performance, device manufacture and quality assurance, and regulatory management with FDA.

2. The Clinical Trials Office of the AFIRM-RCCC at Case Western Reserve University for trial design, data safety monitoring, and regulatory compliance of the study with standards required by the USAMRMC and AFIRM.

3. The Institute for Surgical Research at the Brooke Army Medical Center at Fort Sam Houston which will be one of two clinical performance sites.

 

Kim Warren, PhD will serve as principal investigator for the project at LWI. Dr. Warren is the Head of Cell Therapy Development at LWI and guides the Process Development and R&D Departments in the Cell Therapy business unit. Together with the Cell Therapy production and regulatory divisions at LWI, Dr. Warren will coordinate the manufacture of the autologous engineered skin device, ESS, and its delivery to the clinical performance sites. She will also serve as liaison to a Clinical Research Organization (CRO) which will be contracted by LWI to generate documentation needed for the study, and track the activities of the CRO.

 

Stanton Gerson, MD serves as the Director of the Center for Stem Cell & Regenerative Medicine at Case Western Reserve University. He has extensive experience is performance of clinical trials with cancer treatments, including stem cell therapies. Dr. Gerson will provide assurances of regulatory compliance, data safety monitoring, and assist with review of the study design and statistical analysis of study data. His Center has successfully conducted numerous clinical trials with full regulatory compliance. The Center will work directly with the CRO on contract from Lonza develop the regulatory documents for the study, the investigators brochure and case report forms for data collection. Participation of Dr. Gerson’s Center provides great confidence that regulatory compliance of the study will be fully satisfactory to AFIRM and FDA.

 

Jane Reese serves as Operations Director of the Center for Stem Cell & Regenerative Medicine at Case Western Reserve University and works closely with Dr. Gerson. She will assist with data monitoring and compliance and coordination of the multi-site clinical trial.

 

Steven Wolf, MD holds positions as Professor of Surgery in the Department of Surgery at the University of Texas Health Sciences Center in San Antonio, and director of clinical research at the USAISR. Dr. Wolf will provide advice on planning of clinical applications of autologous engineered skin substitutes at the burn unit at the USAISR. His experience and guidance will greatly facilitate the prospective availability of the investigative therapy for wounded soldiers.

 

Howard Schrayer is an independent consultant to the medical device industry, with more than 25 years experience. His consulting activities are focused on formulating regulatory strategies for medical devices. Mr. Schrayer will serve as a regulatory consultant for this project. His approximate 5% effort contribution is funded by RCCC-AFIRM.

 

Steven Boyce, PhD is the inventor of the ESS technology, and will serve as a technical consultant to the project up to two days per month (10% time). He holds positions as Professor in the Departments of Surgery and Biomedical Engineering at the University of Cincinnati, and directs the Engineered Skin Laboratories at the Cincinnati Shriners Burns Hospital. He will be available to advise on the development of the IDE application, study documents, and will assist with recruitment of a second study site. In addition, Dr. Boyce will continue to provide technical advice for the manufacture of ESS, design of shipping materials and logistics, and training of staff at the study sites.

 

Personnel – Staff

 

Three staff positions are requested at 25% FTE.

46

 

Production Manager – Mark Tracy: will oversee the production staff and operations for the production of the engineered skin devices for qualification runs and clinical trial. Mark and his staff are responsible for carrying out the production process including isolation, expansion, seeding and harvest. Mark will also oversee completion of MBRs, cGMP documentation, and in-process testing. Mark has 6 years experience in cell therapy production at LWI, including 4 years in a supervisory role.

 

Quality Control Manager – John Semon: will oversee release testing of the engineered skin devices for qualification runs and clinical trial. John and his staff are responsible for carrying out the bioburden, sterility, surface electrical capacitance and histology tests. John has been a Quality Control Manager for more than 10 years.

 

Quality Assurance Manager – Vicki Meckley: will oversee quality assurance of production activities, QA audits, controlled documentation and release of engineered skin devices for qualification runs and clinical trial. Vicki has been managed the Quality Assurance team for 4 years and has approximately 10 years experience in the field of biotechnology.

 

Device Manufacture

 

The ESS will be relatively expensive to produce for this initial clinical study from LWI. It is planned that the study will require approximately 4m2 (40,000 cm2) with a cost of goods of $27.47/cm2 for an estimated total cost of $1,098,800; $889,552 of which is charged to the grant budget. The remainder of the cost is a Lonza in-kind contribution. This amount of ESS is required to provide between 0.36 and 0.42 m2 (3,600 - 4,200 cm2) for each of the 10 prospective subjects in the study. This amount per study subject is needed to demonstrate the primary medical benefit which is conservation of donor skin. The study design would propose to manufacture the ESS from a biopsy of split-thickness of an approximate area of 30-40 cm2 for an approximate expansion of the biopsy area by 100 fold. This expansion will demonstrate the primary medical benefit of the device which is reduction of skin harvesting to complete wound closure.

 

Travel

 

Travel costs are requested for the study sponsor (LWI; $15,000), and the associated participants ($10,000). Sponsor related travel will include pre-study visits to the performance sites, training visits, and study closure visits for two staff members. Travel for associated participants will include two on-site visits with the sponsor by Dr. Gerson, Ms. Reese, and Dr. Boyce.

 

Subcontracts:

 

A contract research organization (CRO) will manage the daily operations of the study ($332,000). The CRO partner will be chosen by Lonza. The budget is estimated based on a quote from a representative CRO.

 

Each clinical site will receive a contract for actual costs to perform the study ($126,724 per site X 2 =$253,448). The budget is based on treating 5 patients per clinical site. Final patient enrollment will depend on patient availability.

 

Contributions in-kind from Lonza:

 

1. Preparation of facilities including certification and inspection.

2. Development of packaging and shipping materials.

3. Comparability study (as needed).

4. Project administration.

5. Salaries of key personnel.

6. Majority of production staff payroll expense.

7. Dr. Steven Boyce consultancy.

8. Intellectual Property expenses

47

 

9. Process development expenses.

10. Site and manufacturing regulatory compliance.

 

Appendix List:

 

1. Letter of collaboration, USAISR

2. Letter of consultation, Steven Boyce, PhD

3. Case Report Forms from previous study

4. Boyce publications

4.1. ASC 2008

4.2. J Trauma 2006

48

 

 

49

 

 

50

 

 

51

 

 

52

 

 

Steven Boyce, Ph.D. Professor Division of Burn Surgery Department of Surgery College of Medicine P.O. Box 670558 Cincinnati OH 45267-0558 Phone: (513) 872-6080 Fax: (513) 872-6107 Email: boycest@uc.edu

 

 

 

23 February 2009

 

Kim Warren, Ph.D.

Lonza Walkersville, Inc. (Lonza)

8830 Biggs Ford Road

Walkersville, MD 21793

 

Re: Proposal for clinical trial of PermaDerm for burns

 

Dear Dr. Warren:

 

I am pleased to serve as a consultant to Lonza’s proposal to perform a clinical trial of PermaDerm, an autologous engineered skin substitute for treatment of life-threatening burns.

 

As ou know, I have extensive expertise in the development of human cell culture systems, skin biology, wound healing, and biocompatability of implantable materials. My research interests include mechanisms of wound healing with engineered human skin, and biologic fidelity of engineered tissues to natural tissues by regulation of cellular phenotypes. My laboratory has developed engineering systems for skin substitutes that are used to treat burn wounds with significant advantages to traditional split thickness skin grafting procedures. These technologies have been translated to clinical investigations, patented, and licensed to Cutanogen Corporation which is a subsidiary of Lonza. As the founder of Cutanogen Corporation, I have an established working relationship Lonza which will facilitate accomplishment of the proposed clinical trial.

 

The study which Lonza proposes is relatively simple in design, but will require both technical advice and close tracking of activities to complete successfully. I shall be available to assist with study design, review of manufacturing procedures, and recruitment of clinical study sites as needed. I shall also be available to act as liaison to other work groups within the RCCC-AFIRM consortium to facilitate performance of the project.

 

There delivery of an autologous engineered skin substitute for catastrophic burn injuries promises to raise the standard of burn care nationwide, and reducing morbidity and mortality in this high-risk population of patients. I look forward to working with Lonza to demonstrate these benefits, and to deliver this important new therapy.

 

I wish you every success with the proposal.

 

Sincerely,

 

/s/ Steven Boyce

Steven Boyce, Ph.D.

53

 

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE# G980023

 

SCHEDULE FOR DATA COLLECTION

 

DATA COLLECTED	Prestudy	CSS Surgery Day 0	POST-OPERATIVE DAY
			7	14	28	91	182	365	Later if possible
Informed Consent	X
Past Medical History	X
Physical Exam	X	X			X	D/C
Serum for Antibodies	X				X
Skin Biopsy for Tracing & Culture	X
Pretreatment of Sites	X
Biopsy & Photo of Recipient Sites		X
Microbial Culture of Recipient Sites	X	X	X	X
Photography		X	X	X	X	X	X	X	X
CSS Tracing In Vitro		X
Study Site Tracings				X	X
% Engraftment				X	X
Healed Area : Donor Area Ratio					X
Qualitative Outcome				X	X	X	X	X	X
Healed Wound Biopsy (as possible)			X	X	X	X	X	X	X
Adverse Event Summary				X	X
Investigator Global Assessment				X	X			X

 

*Primary data analysis will be performed on post-operative day 28, and as close as possible to day 365.

# Punch biopsies (3mm) will be obtained at scheduled time points, not to exceed a total of 6 samples nor 5% of the healed graft.

54

 

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE# G980023

 

Registration Form

 

Patient Initials:

Study Registration Number:

CSS

Hospital ID#:

Gender:

M / F

Birthdate:

Age:

Race:

M / D / Y

Burn Date:

Admission Date:

M / D / Y

M / D / Y

 

TSBA (m2, from			% burned:			% 2º:			% 3º:
nomogram):

 

 

Allergies: _____________________________________________________________________

 

Inclusion - Must answer "Yes" to the following:

 

Yes		No
				Age is birth - 75 years
				TBSA $50% (or $10% full thickness)
				Burn includes full thickness
				Patient is not showing signs of sepsis
				Expected to need grafting after 3 weeks post primary biopsy
				Informed Consent signed

 

Exclusion - Must answer "No" to the following:

 

Yes		No
				Patient is pregnant or lactating
				Patient is a prisoner
				Patient is mentally incompetent

 

Date enrolled: __________

M / D / Y

55

 

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

 

 

Patient Initials:			Date:
Study Registration Number:	C   S  S		Study Day:	Prestudy

 

PAST MEDICAL HISTORY

 

	No	Yes	Comments
HEENT
Respiratory
Cardiovascular
Gastrointestinal
Genitourinary
Musculoskeletal
Neurological
Endocrine
Substance Abuse
Psychological
Other

 

Form CSS 7.3

Revised: October 2003

56

 

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

 

Patient Initials:			Date:
Study Registration Number:	C   S  S		Study Day:

 

PHYSICAL EXAM

 

 

		Admission
		Pre-surgery
		POD 28
		Discharge

 

	Normal	Abnormal	Comments
HEENT
Respiratory
Cardiac
Circulation check
Gastrointestinal
Genitourinary
Musculoskeletal
Neurological
Endocrine
Integumentary

 

Serum for circulating antibodies obtained:

 

	Prestudy
		Date
	Study Day #28
		Date

 

Form CSS 7.4

Revised: October 2003

57

 

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

 

Burn Estimate and Diagram			Burn Diagram
Age vs. Area
Initial Evaluation
Pt. Initials:
Pt. Reg. No.:
Investigator
Completing Burn Diagram
	Color Code
	Red - 3º
	Blue - 2º

 

 

Area	Birth 1 yr.	1-4 Yrs.	5-9 Yrs.	10-14 Yrs.	15 Yrs	Adult	2º	3º	Total	Donor Areas
Head	19	17	13	11	9	7
Neck	2	2	2	2	2	2
Ant. Trunk	13	13	13	13	13	13
Post. Truck	13	13	13	13	13	13
R. Buttock	2 ½	2 ½	2 ½	2 ½	2 ½	2 ½
L. Buttock	2 ½	2 ½	2 ½	2 ½	2 ½	2 ½
Genitalia	1	1	1	1	1	1
R.U. Arm	4	4	4	4	4	4
L.U. Arm	4	4	4	4	4	4
R.L. Arm	3	3	3	3	3	3
L.L. Arm	3	3	3	3	3	3
R. Hand	2 ½	2 ½	2 ½	2 ½	2 ½	2 ½
L. Hand	2 ½	2 ½	2 ½	2 ½	2 ½	2 ½
R. Thigh	5 ½	6 ½	8	8 ½	9	9 ½
L. Thigh	5 ½	6 ½	8	8 ½	9	9 ½
R. Leg	5	5	5 ½	6	6 ½	7
L. Leg	5	5	5 ½	6	6 ½	7
R. Foot	3 ½	3 ½	3 ½	3 ½	3 ½	3 ½
L. Foot	3 ½	3 ½	3 ½	3 ½	3 ½	3 ½
						Total:

 

Shriners Hospital for Children

Cincinnati Burns Institute

Burn Diagram

 

Form CSS 7.5

Revised: October 2003

58

 

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

 

Patient Initials:			Date:
Study Registration Number:	C S S		Study Day:	Prestudy

 

PRESTUDY DATA
Primary Biopsy for Skin Cell Cultured obtained								Date
								PBD
					from			Donor site
Parameter			Date			Site A		Site B
						AG	CSS	AG	CSS
Excision of Eschar
Temporary wound coverage
achieved with:
Organisms Isolated
(only one pre-application
Microbiology culture needed)
Pretreatment Topicals Used:
Blood Culture		Organism Isolated		Treatment Meds			Systemic Treatment (dates)

 

Form CSS 7.6

Revised: October 2003

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

 

 

Patient Initials:			Date:
Study Registration Number:	C S S		Study Day:	#0

 

 

 

 

 

 

Recipient Wound Bed
	Pre-Application Data					Post-Application Data
Study Site A B	Temporary Cover Excised   _/_/_ (Date)   _/_ /_ (Date)	Punch Biopsy Taken		Microbial Culture Taken _ Y _ N   _ Y _ N	Photos Taken _ Y _ N   _ Y _ N	Type & Size of Grafts Applied
		_  Y _  N
		_  Y _  N
			Site Identification
Study Site   A	Location		Landmarks
			From ( ) to the center of Study Site measures ( )
			From ( ) to the center of Study Site measures ( )
B			From ( ) to the center of Study Site measures ( )
			From ( ) to the center of Study Site measures ( )

59

 

CULTURED SKIN SUBSTITUTE

Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

 

Patient initials: __________________

Patient registration number: CSS_________

 

ADVERSE MEDICAL EVENTS

List below any new event(s) or changes in the status of previously recorded event(s) including cessation of event(s)

Instructions:   Frequency: Check if constant or place a number that best describes frequency in the per day column.   Duration: If not consistent, indicate the duration per episode. Place a number in box that best describes duration.	Date Started M/D/Y	C O N T I N U I N G	Date Stopped M/D/Y	Frequency		Duration			Relationship to Test Materials If unlikely, or not test material related, indicate probable cause	Action(s) Taken   (List as many as apply)	O U T C O M E
				C O N S T A N T	Per Day	M I N U T E S	HOURS	DAY
EVENTS  None

 

Keys:

SEVERITY   1. Mild 2. Moderate 3. Severe 4. Life-threatening or intolerable 5. Fatal

Relationship to test material: 1. Not related 2. Unlikely related 3. Possibly related 4. Probably related 5. Definitely related

Action(s) Taken: 1. None 2. Test Material Dosage Reduced 3. Test Material Administration Interrupted, Reinstated at Same Dose 4. Test Material Administration Interrupted, Reinstated at Lower Dose 5. Test Material Discontinued 6. Medication Administered 7. Treatment other than Medication Administered - Specify* 8. Laboratory or other Diagnostic Test(s) Done 9. Randomization code broken. Date __/__/__.

Outcome: 1. Recovered with treatment 2. Recovered without treatment 3. Alive with sequelae 4. Event is continuing and controlled with treatment 5. Event is continuing without treatment 6. Patient died 7. Unknown

 

— Record in Concomitant Medications section of Case Report form

* Record in Comments below

^ Note - REPORT TO FDA and IRB, IF MEDICAL EVENT IS UNEXPECTED, SEVERE,

LIFE THREATENING OR FATAL

 

Comments:							__/__/__
				Investigator

 

Form CSS 7.8

Revised: October 2003

60

 

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

 

Patient Initials			Date:
Pt. Reg. No.	C S S		Study Day:

 

 

Qualitative Outcome

Site B

____ % healed ____ % healed

____ % open ____ % open

 

(The following scores will reflect only those areas which are healed.)

*Required Study Day: #14, #28, and subsequent clinic visits.

 

 

Parameter

Site A score

Site B score

Percentage: 1) Erythema

___

___

___

___

___

___

___

___

0

1

2

3

0

1

2

3

normal

pink

red

purple

normal

pink

red

purple

Percentage: 2) Pigmentation

___

___

___

___

___

___

___

___

0

1

2

3

0

1

2

3

none

hypo

normal

hyper

none

hypo

normal

hyper

Percentage: 3) Skin Pliability

___

___

___

___

___

___

___

___

___

___

___

___

0

1

2

3

4

5

0

1

2

3

4

5

Normal

Supple

Yielding

Firm

Rope

Contract

Normal

Supple

Yielding

Firm

Rope

Contract

Percentage: 4) Scar Height

___

___

___

___

___

___

___

___

0

1

2

3

0

1

2

3

Flat

< 2mm

> 2mm

> 5mm

Flat

< 2mm

> 2mm

> 5mm

5) Site regrafting: (If partial or total, see below)

Total

Partial

None

Total

Partial

None

 

SITE RECONSTRUCTION

 

	Site A	Site B
Surgery date Study day # Procedure % CSS remaining Was CSS excised?

 

61

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

 

Patient Initials			Date:
Pt. Reg. No.	C S S		Study Day #

 

 

Investigator's Global Assessment at POD 14

 

1) Percentage healed graft (from tracing): Site A: ________ Site B: ________

A) Cause(s) of failure of auto-CSS:

________________________

________________________

B) Is regrafting needed: Yes  No 

C) Date that the manufacturer was notified of device failure:

D) Enter the date of the Monday of the week in which regrafting is expected to be performed: _____________________________________

E) State the type of graft that is expected to be used: auto-CSS  skin autograft 

State reason for choice: _____________________________________

 

2) Healed area: donor area ratio for cultured skin substitute?

 

3) Were there any adverse events during the course of this study?

 

No ___ Yes ___ (see adverse event record)

 

4) Status of subject

 

___ Continues in study

___ Discontinued from study (see number 5)

 

5) If subject was discontinued from the study, indicate reason:

 

___ graft failure

___ adverse event

___ subject request

___ protocol violation

___ other, please specify _____________________________________

 

6) Was the subject discharged with cultured skin intact?

 

Yes ___ No ___ Not yet discharged ___ Discharge date

 

To Be Completed and Signed by Investigator

			Date Signed:
Principal Investigator

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

Investigator's Global Assessment at POD 28

1) Percentage healed graft (from tracing): Site A ___ Site B ___

2) Healed area: donor area ratio for cultured skin substitute?

_____________________________

3) Were there any adverse events during the course of this study?

No ___ Yes ___ (see adverse event record)

4) Status of subject

___ Continues in study

___ Discontinued from study (see number 5)

5) If subject was discontinued from the study, indicate reason:

___ graft failure

___ adverse event

___ subject request

___ protocol violation

___ other, please specify _____________________________________

6) Was the subject discharged with cultured skin intact?

Yes ___ No ___ Not yet discharged ___ Discharge date

To Be Completed and Signed by Investigator

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

* Punch biopsies will be obtained at scheduled time points, not to exceed a total of 6 healed wound samples nor 5% of the healed graft.

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

Microbiology

*Note- (S) Swab or (Q) Quantitative

(Required Study Day #0, #7, #14)

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

Blood Cultures

Systemic Antibiotic Therapy

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

Photography Log

Form CSS 7.15

Revised: October 2003

CULTURED SKIN SUBSTITUTE

IRB Protocol #95-7-26-1 (autologous CSS)

IDE # G980023

Site Regrafting Log

POD, post-operative day

Form CSS 7.16

Revised: October 2003

The Journal of TRAUMA Injury, Infection, and Critical Care

Cultured Skin Substitutes Reduce Requirements for Harvesting of Skin Autograft for Closure of Excised Full-Thickness Burns

Steven T. Boyce, PhD, Richard J. Kagan, MD, David G. Greenhalgh, MD, PetraWarner, MD, Kevin P. Yakuboff, MD, TinaPalmieri, MD, and Glenn D. Warden, MD

The Journal of TRAUMA Injury, Infection, and Critical Care

Table 1 Enrollment and Treatment Data

Cultured Skin Reduces Autograft Harvesting

The Journal of TRAUMA Injury, Infection, and Critical Care

I to 4 was expanded and stapled to wounds. CSS and AG were dressed with fine mesh gauze, and covered with bulky gauze containing perforated red rubber catheters that were secured with Spandex (De Royal, Pmvell, TN) that was stretched to apply gentle pressure and to immobilize the grafted sites. If CSS and AG were LUlller the same dressing, the irrigatiun solutiun fur CSS was used. Postoperatively, CSS were irrigated with the solution of nutrients and antimicrobials described above, at a dosage of 1 mL per c.m2 CSS three times per day for 5 to 7 days. Dressing changes for CSS and AG were routinely performed on postoperative days (POD) 2 and 5, and all staples and N-terface were removed on POD 5. CSS were treated with an ointment (NBN) consisting of equal parts NeoSporin (Pfizer; New York, NY), Bactroban (GlaxoSmithKline; London, UK), and Nystatin (Wyeth Pharmaceuticals; Madison. NJ), and covered with dry bulky gauze. Dry, keratinized areas were treated with Curci (Kao Brands Co .• Cinciru1ati, OH) lotion and wet areas were treated with NBN ointment on Adaptic (Johnson & Johnson; New Bruswick, NJ) until healing was complete. Daily dressing changes were performed from POD 6 to 7, after which dressings were changed t\.vice daily. If healing was not complete by POD 15, routine wound care for AG was performed on CSS sites. AG was usually irrigated for 5 days with alternating solutions of 5% wt/vol mafenide acetate in water and a solution of 40 J.Lg/mL neomycin and 700 U/mL polymyxin B in saline. Dry dressings for AG routinely consisted of Adaptic coated with an ointment consisting of either 3 parts bacitracin and 1 part silver sulfadiazine if no yeast species were cultured from the grafts, or equal parts silver sulfadiazine, bacitracin, and nystatin if yeast were cultured. Statistical Analvsis Primary analyses of data were performed on POD 28 for quantitative and qualitative endpoints. and at 1 year ± 1 month for qualitative outcome. Qualitative data sets \vcre analyzcd for ovcrall significance by the Kruskal-Wallis test. If overall significance was found. then differences were subjected to Wilcoxon's rank sum test. Data from positive! negative scoring of site regrafting was subjected to Fischer's exact test. For the endpoint, ratio of closed-to-donor areas, which is defined in the IDE protocol as the primary medical benefit, a statistical power analysis was pertormed based on preliminary data. For an alpha value of 0.05, and beta values ranging from 0.95 to 0.80, tbe estimated size ofthe population to detem1ine a statistical difference between the CSS and AG treatments was 13-21 patients. For that endpoint. a single-value I test was applied to compare CSS to a maximum value of 4 per harvest of AG. Values for expansion of AG were most often less than 3, but were not recorded for all AG applied to all patients in this study. Tllis statistical approach minimizes the benefit of CSS for tllis endpoirit, and therefore was considered the most conservative statistical analysis.	RESULTS Figure I shows microscopic anatomy of AG (left panel) and CSS (right panel) before grafting. Both have dermal and epidermal components with a total thickness of less than 400 J.Lm. The dermal component of CSS consists of reticulations of collagen-glycosaminoglycan (GAG) biopolymer populated with cultured fibroblasts to which the epidermal component is attached biologically. The epidermal component consists of cultured keratinocytes that stratify and differentiate to form an analog of stratum corneum, which is a precursor of functional epidermal barrier. The CSS generally resemble the anatomy of splitthickness skin but lack blood vessels. Therefore, CSS develop vascular perfusion entirely by angiogenesis, rather than by inosculation of blood vessels in the wound to those in the graft as occurs in AG. Most CSS in this study were approximately 6 X 6 cm in area, but a limited number of CSS of larger area (-12 X 12 cm) were also applied. Surgical application and healing during the first year after surgery are shown in Figure 2. Not sUllJrisingly, it was found that overlapping of the edges of CSS suppressed formation of granulation tissue between grafts and reduced linear scars after healing. In this patient, CSS were applied over excised, full-thickness burns and accomplished more than 90% wound closure at POD 14. At POD 28, the closed wounds are stable and use of pressure garments was begun. The healed CSS was stable, pliable, and hypopigmented at POD 69 and remained pliable and hypopigmented at POD 479. By 1 year after grafting in this patient, the autograft had developed greater areas of hypertrophic scar than the CSS. Epithelial engraftment and wound closure at POD 14 (Fig. 3A) was 81.5% tor CSS compared with 94.7%, tor AU which was statistically significant (p < 0.05). A need tor regrafting of CSS (13 of 40) was observed, but not for AG (0 of 40). Nonetheless, the magnitude of regrafting was usually small and the quantitative closure of wounds was much greater for CSS than AU (Fig. 38). The ratio of closed wound area to donor skin area tor CSS was 66.2 versus a maximum of 4 per harvest of AG. This difference is highly significant (p < 0.0 I), represents a reduction of donor skin harvesting of more than an order of magnitude by use of CSS, and detines the medical benefit or this alternative therapy for burn pa-
	Fig. 1. Histologic anatomy of split-thickness skin and cultured skin substitutes before Surgery. (A) Split-lhickness skin has a fully keratinized epidermis and vascularized dermis. (B) Cultured skin substitute has partial keratinized epidermis and dermal substitute without a vascular network. Scale bar = 0.1 mm.

Cultured Skin Reduces Autograft Harvesting

	Fig. 3. Engraftment and donor skin reduction. (A) Percentage areas (mean ± SEM) closed at POD 14 were 81.5 ± 2.1 for CSS and 94.7 ± 2.0 for A G. (B) Ratios of closed-to-donor areas at POD 28 were 66.2 ± 7.6 for CSS and 4.0 ± 0.0 for A G.
	cases, emphasizing the therapeutic impact of this device in life-threatening burns. On averagc, ess covered 20.3% TBSA, and AG covered 52.5% (Fig. 4B).   No differences in qualitative outcome between CSS and AG were found at I year after grafting (Fig. 5) according to ordinal scoring by the Vancouver Scale for scar assessment. Significant differences were found between ess and AG at time points of 6 months and earlier. Application of ess without the expanded mesh of AG generated a smoother surface and CSS were consistently hypopigmented. Both of these factors contributed to lower scores for ess before 6 months. Histologic anatomy of ess and AG is shown in figure 6. At 5 months after grafting, the epidermis had matured and remaincd stable and tightly-adhcred to conncetive tisslle. Neither healed AG (Fig. 6A) nor ess (Fig. 6B) developed glands or follicles. Vascularity had decreased and collagen distribution was orthogonal, not linear as in scar. The epidermal surface and dermal-epidermal junction remained relatively linear indicating the absence of rete peg formation. At 15 months after grafting, AG (Fig. 6C) had developed a well interdigitated dermal-epidermal junction, and CSS (Fig. 6D) showed a nonlinear busement membranc zone.
Fig. 2. Clinical observation and healing during the first postoperative year. (A) Surgical application of large (.wlid box) and small (dotted box) formats of cultured skin substitutes (CSS). (B) Postoperative day (POD) 14. (C) POD 28 shml's areas treated with split-thickness autograft or CSS. (D) POD 69. (E) POD 479. Scales in mm.
tients. This is the primary benefit that is reported to the US FDA for measurement of the efficacy of this device. A positive correlation was found between the percent TBSA of wound closure with ess at POD 28, and the percent TBSA full-thickness burn (Fig. 4A). Importantly, the range of percent TBSA closed extended to 60% or greater in selected	Fin. 4. Correlation of percent total body surface area (TBSA full-thickness burn and percent TBSA closed. (A) Positive correlation (r2 = 0.37. p < 0.0001) was detected between percent TBSA closed with CSS and percent TBSA burned. (B) TBSAs (mean ± SEM) closed at POD 28 were 20.3 ± 2.0 for CSS and 52.5 ±2.0 for AG

The Journal of TRAUMA Injury, Infection, and Critical Care

Cultured Skin Reduces Autograft Harvesting

The Journal of TRAUMA Injury, Infection, and Critical Care