Development of a bench-scale bioreactor for the fabrication of a tissue engineered skin construct using a PLGA scaffold

ABSTRACTThis report details the production and design of a bioreactor capable of fabricating a skin replacement construct was successful. Testing of the bioreactor protocol indicated that the design of the system promoted good mixing, as indicated by seven days of healthy cell growth. Also, adherence to the polymer scaffold was observed within twenty-four hours subsequent to the seeding of NIH 3T3 fibroblast cells. This design may be expanded if a larger time scale was made available.

INTRODUCTIONMore than two million burn injuries occur each year in the US, resulting in 100,000 to 300,000 hospitalizations.1 The development of tissue engineered skin is quickly becoming a major form of treatment for the reconstruction of second and third degree burns. The two main concerns regarding the use of artificial skin are a 10 to 14 day waiting period for maturation of the skin construct, and the prevention of infection in the patient by decreasing wound exposure. To address these major concerns, we have devised a method of creating readily available sources of skin constructs.

Therefore, our primary goal is to produce a novel living skin substitute containing both dermal and epidermal layers on a biodegradable polymer scaffold in a continuous perfusion bioreactor. Previously, the development of a single-pass perfusion reactor system for in vitro growth of a three-dimensional human dermal replacement was accomplished by Halberstadt et al.2 Our objective is to provide plans for scale up of the production process for the bioartifical skin that can compete with other products currently on the market by supplementing the construct with an epidermal layer composed of keratinocytes, creating a recycle line for the bioreactor, and potentially adding growth factors to the media. It is expected that our revised system will produce better cell adherence. The addition of the epidermal layer will make the skin replacement more closely resemble actual human skin in both form and function, and provide the protective covering of keratin. The recycle line will aid in seeding the mesh with cells and potentially increase growth rates while reducing media requirements.

Some of the competing products currently on the market are Dermagraft® (Advanced Tissue Sciences, Inc.), TransCyteTM (Advanced Tissue Sciences, Inc.), Integra® (Integra Life Sciences), and Apligraf® (Organogenesis Inc.). Dermagraft® is produced by seeding one layer of dermal fibroblasts onto a three-dimensional scaffold consisting of a bioabsorbable material. The cells grow and divide, producing collagens, extracellular matrix proteins and growth factors found in normal, healthy human dermis. In TransCyte®, fibroblasts are grown on a mesh, where they divide and produce growth factors, collagens and other proteins to form a functioning human dermis. The fibroblasts are lysed, and then frozen for shipment and stored for off-the-shelf use.3

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Integra® is a composite skin graft made of collagen based dermal lattice with an outer silicone covering. As the dermal component is slowly degraded, the silastic sheet is eventually removed and covered with an autograft.4 Apligraf® is manufactured by initially seeding the polymer with allogeneic fibroblasts to form the lower dermal layer of the construct. As the fibroblasts move through the collagen, the dermal layer is rearranged, resulting in the production of more collagen. Then, keratinocytes are placed on top of dermal layer, forming the upper epidermal layer. The epidermal layer is exposed to the air, which prompts the formation of the outer protective layer of skin (stratum corneum).5

LITERATURE REVIEWPLGA Application as a Polymer ScaffoldPLGA, poly(lactide-co-glycolide), is biodegradable and biocompatible. Halberstadt et al. describe the development of a single-pass perfusion reactor system for in vitro growth of a three-dimensional human dermal replacement. In their system, human neonatal dermal fibroblasts are grown on a biodegradable, PLGA mesh (Vicryl; Ethicon).3 Also, PLGA is widely used for many medical applications. Furthermore, the metabolites it generates as it degrades are relatively harmless.6,7

Mesh versus Knitted Polymer In this study, Cooper et al. found that the knitted mesh did not roll up as much as the woven mesh when cells were placed on it or when it was in solution, thus making it easier to handle. In full-thickness, open skin wounds in athymic mice, a more open knit or weave was shown to enhance take and vascularization compared to a denser or tighter weave. The Vicryl knitted mesh used in their study was the same mesh used in the current investigation.8

Perfused Versus Static Cell CultureHalberstadt et al. demonstrated that it took 6 fewer days to grow a certain amount of fibroblast cells on Vicryl mesh in a single-pass perfusion process (16 days) compared to a static process (22 days). In the current study, new goals were to increase the growth rate and to decrease media requirements while maintaining growth rates by the addition of a recycle line to the continuous perfusion bioreactor.7

MATERIALS AND METHODSBioreactor DescriptionThe bioreactor consisted of an untreated, petri-style, round polystyrene dish that was chemically sterilized by filling it with ethanol and placing it in the vacuum hood overnight. The dish was equipped with a loose fitting lid, and was 150mm in diameter and 25mm tall. The lid had a 8 x 8 cm window removed and was replaced with Breathe EasyTM, a breathable, sealing polyurethane membrane. This allowed sterile gas exchange of O2, CO2, and water vapor into the reactor from the surrounding environment in the incubator. Three ports were drilled into each side of the dish to allow for 2 inlet lines, 2 outlet lines, and a recycle line on each side. The inlet and outlet tubing was Pharmed ® tubing with an inner diameter of 0.8mm and an outer diameter of 4mm. The recycle line tubing was slightly bigger with an inner diameter of 1.6mm and an outer diameter of 4.8mm.

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The various lengths of tubing were connected with polypropylene luer lock connectors reinforced with Teflon® tape (PTFE). The inlet line was split with a T-connector just before entering the bioreactor, thus providing two separate inlet jets to encourage mixing. Two outlet lines left the bioreactor and were joined by a T-connecter before entering the pump. The recycle line was one continuous closed loop starting and ending at different sides of the bioreactor. Figure 1 shows the schematic diagram showing the placement of the inlet, outlet, and recycle lines on an overhead view of the bioreactor.

Figure 1. A schematic of the bioreactor, including ports, recycle stream, and waste lines.

Figure 2 shows an actual picture of the bioreactor without the polymer mesh and lid. The ruler at the bottom of the picture is approximately 18 cm long.

Figure 2. A picture of bioreactor protocol.

The preceding figure also shows the outline of the Vicryl ® mesh and its supports. The Vicryl ® knitted mesh was 7.6cm x 7.6cm square and had a support post located at each

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corner. The stainless steel supports were secured to the bottom of the bioreactor by applying Dow Corning Medical Adhesive glue to their round bases. Then, two immobile round disks placed above and below the mesh on the posts suspended the mesh above the bottom of the bioreactor, thus allowing flow to circulate above and below the mesh.

Figure 3. A schematic of the top and side views of the polymer support.

The Vicryl ® knitted mesh, purchased from Ethicon and composed of poly(lactide-co-glycolide) or PLGA copolymer, with lactide and glycolide derived from lactic acid and glycolic acid, respectively. The mesh was 0.19 mm thick and the pore size was approximately 5mm x 5mm. According to product information, the mesh had an initial average burst strength of 63 lbf.

As shown below in Figure 4, glycolic acid (HOCH2COOH), reacts with itself to form glycolide dimmer. Also, lactic acid (HOCH(CH3)CH2COOH), also reacts with itself to form lactide dimmer. The product, PGLA, then contains the desired properties of the lactide and glycolide.

Figure 4. Synthesis of poly(lactide-co-glycolide).11

Three Watson-Marlow 101U/R –MK2 peristaltic pumps powered all flow. These pumps had settings from 1-99, and the flow rates associated with the settings were characterized by experiments detailed in appendix C. Each line (inlet, outlet, and recycle) had its own pump, and the figure below depicts the general placement of each along with a side view of the bioreactor and lid.

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Figure 5. A diagram of the entire system: the bioreactor, pumps, and containers

Cell Culture of Fibroblasts and KeratinocytesIn order to produce the large quantities of cells required for seeding the PLGA mesh, standard mammalian cell culture techniques were employed. Mouse fibroblasts (NIH/3T3) were obtained from the Department of Chemical Engineering at Iowa State University. Mouse keratinocytes (K213) were obtained from the Skin Diseases Research Center, Department of Dermatology at Yale University School of Medicine. Each cell line was handled independently of the other to minimize the risk of cross-contamination. However, the procedures and materials used were identical for both cell lines.

Cells were revived immediately from cryostorage in liquid nitrogen, into tissue culture flasks (T-25, Corning) using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with: 10% Fetal Bovine Serum (FBS), 50 mg/mL vitamin C, 5 mg/mL insulin, antibiotics (50 g/mL penicillin G, 50 g/mL Streptomycin, 100 g/mL Neomycin) and buffered with sodium bicarbonate. Media was sterilized by vacuum filtration (.22 m cellulose acetate, Corning). This media formulation (subsequently, DMEM) was the standard used throughout all experiments. Manual manipulations of culture vessels were performed in a sterile vertical laminar flow safety cabinet to minimize the risk of contamination via aerosols and airborne microorganisms. During growth, cell culture flasks were maintained in humidified incubators at 37 degrees C, with a 5% carbon dioxide atmosphere for buffering. Cell growth and viability were monitored using an inverted, phase contrast light microscope (C-10, Olympus). Both K213 mouse keratinocytes and NIH/3T3 mouse fibroblasts are anchorage-dependent cell lines, requiring adherence to a substrate in order to propagate. In vitro viability was assessed by degree of adherence and cell morphology.9

After cells reached confluency in the T-25 (25 cm2 surface area) tissue culture flasks, they were passaged to larger T-75 (75 cm2 surface area) and T-225 (225 cm2 surface area) tissue culture flasks (Corning). Cell passaging was accomplished by washing the cells with Hank’s Balanced Salt Solution (HBSS) followed by a wash of 0.25% trypsin and ethylenediaminetetraacetic acid (EDTA) in HBSS and subsequent addition of DMEM to dilute the cells to the desired seeding ratios.10

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Growth Factor ExperimentIn order to quantify the effect of growth factors upon the growth rate of fibroblast cells, an in vitro cell culture experiment was performed. Mouse fibroblasts (NIH/3T3) were seeded at a density of approximately in six-well tissue culture plates (Corning). DMEM was prepared for the control case, and then supplemented with appropriate levels of growth factors for each experimental case, as indicated in Figure 6. Each case was performed in triplicate

Experiment ID Description Growth Factor Concentration (ng/mL)

1 Control 02 bFGF (High) 203 bFGF (Low) 54 EGF (High) 205 EGF (Low) 56 bFGF/EGF (1:1) 10 : 107 bFGF/EGF (1:4) 4 : 168 bFGF/EGF (4:1) 16 : 4Figure 6 Experimental cases for growth factor cell culture experiment.

Cells were harvested from each well via trypsinization and counted using a hemocytometer according to the technique recommended by Freshney.9 Cell count data was collected and analyzed for statistically significant differences between control and experimental groups.

Growth Factor Concentration DeterminationIn order to ascertain the optimum concentration of Epidermal Growth Factor (EGF) to Basic Fibroblast Growth Factor (bFGF), eight experiments were designed to determine the individual and combined effects of the growth factors. First, the effect of each individual growth factor was tested, using high (20 ng/ml) and low (5 ng/ml) concentrations. Then, the combined effects of the growth factors were tested using four-to-one (16:4 ng/mL), one-to-four (4:14 ng/mL) and one-to-one ratios (10 ng/mL). Each experiment was done in triplicate, compared to a control, and the effects were measured by variable cell count. Flow ExperimentsWe created a bioreactor prototype in order to characterize flow patterns throughout the reactor. This prototype was identical in size to the final bioreactor design, but it did not contain any polymer mesh. We also used the larger tubing to achieve a better developed flow, thus reaching steady state faster.

First, the bioreactor and all tubing were filled with water. All three pumps were set at the same flow rate of 0.09 mL/s and allowed to run for a few minutes to ensure that the bioreactor was at or near steady state. At this point the inlet fluid was changed to the blue tracer fluid. After the blue tracer fluid had filled the reactor, the recycle line was hooked up to a tank of red fluid to better show the recycle flow patterns.

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RESULTSThe following figures show that our idea of dual inlet and outlet jets promoted mixing and did an excellent job of directing fresh incoming media towards the area of the polymer mesh. The recycle line served the purpose of further channeling media across the reactor channel. In addition, some stagnation near the walls of the bioreactor can be seen in later figures. We feel this is minimized by the circular design and would only be exaggerated by a square or rectangular bioreactor.

Figure 7: The blue tracer fluid has just entered the bioreactor. Note that the bottom inlet line was not flowing due to a trapped air bubble.

Figure 8: Mixing continues and the bottom line starts to flow.

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Figure 9: The inlet jets are directed almost completely towards the two left-hand posts, providing good media circulation near the mesh site.

Figure 10: The recycle inlet stream makes a visible path of clearer fluid in the center of the reactor, thus encouraging circulation and turbulent mixing in the bioreactor. The blue fluid first reaches the far right side of the bioreactor in this figure.

Figure 11: All fluid in the bioreactor is tinted blue. Notice the lighter band of fluid around the outside of the reactor, indicating some stagnation on this outside ring.

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Figure 12: This picture shows the start of the colored recycle fluid. The recycle inlet has just been disconnected and reconnected to a bottle of red fluid. Note that the recycle outlet is not running at this time.

Figure 13: The red recycle stream becomes more prominent. Because the recycle outlet is not hooked up, the red liquid has trouble flowing across the bioreactor.

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Figure 14: The recycle outlet is again hooked up. The recycle stream is immediately being pulled more quickly across the bioreactor.

Pump Flow Rate CharacterizationThe Watson Marlow peristaltic pumps had flow rate settings from 1-99, and the volume of liquid pumped in a given time was dependant both on the flow setting and the tube size. We performed experiments in the lab in order to determine the relationship between both variables.

Figure 15 shows the volumes of liquid pumped in a given time. The first number in the key depicts the size of tubing used (1 for the larger Pharmed ® tubing with an ID of 1.6mm and 2 for the smaller Pharmed tubing with an ID of 0.8mm). The second number in the key depicts the pump’s flow rate setting.

Figure 15: Pump Flow Data: Volumes pumped in a given amount of time by tube size and pump setting.

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Linear regression gave the slopes, or volumetric flow rates, for each combination of tube size and pump setting. These results are shown in the following table:

Tubing SettingFlow rate (mL/s)

1 95 0.08881 50 0.04971 25 0.03052 95 0.02972 50 0.01602 25 0.0081

Figure 16. Results from the linear regression of flow rates.

Plotting the flow rates vs. the pump settings gave almost perfectly linear correlations, thus giving us two relationships (one for each tubing size) between the pump setting and the resulting flow rate. (see Figure 17) These equations made it easy for us to calculate the number of media changes per day that would result from the specific flow settings.

Figure 17: Flow Rates vs. Pump Settings: The graph clearly shows the linear relationship between the peristaltic pump setting and the resulting flow rate.

Growth of skin replacement in bioreactorThe growth of the fibroblast cells on the mesh was first evaluated on Day 1 (24 hours after seeding). During the 24 hours after seeding, only the recycle pump had been running. The bioreactor was removed from the incubator and placed in the laminar flow hood where all flow lines were disconnected. Sterile luer lock plugs were placed on the lines attached to the bioreactor to maintain sterile conditions in the reactor while viewing under the inverted, phase-contrast microscope. Cells attached to the bottom of the reactor on the polyurethane membrane and looked healthy. There were very little cells attached to the mesh. A small leak on the inlet recycle line was also discovered. Due to the leak and the ineffectiveness of the polyurethane membrane for preventing cell attachment, the mesh was removed from the bioreactor and placed on a polyethylene film in a new

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circular polystyrene dish, similar to the original bioreactor but without flow lines, post supports, or the polyurethane membrane on the bottom of the dish. Cells were passaged from the original bioreactor and re-seeded on the mesh in the new dish. The new dish was then placed back in the incubator and cells were allowed to grow in static culture for 24 hours.

The mesh was examined again on Day 2 and results were similar to those obtained on Day 1. Cells had attached to the polyethylene film and areas of the dish not covered with the film, and a few had attached to the mesh. The mesh was then relocated to a third dish containing approximately 40 mL of 20% w/w poly(vinyl alcohol) (8,000 MW) in dH2O. No glutaraldehyde was added to cross-link the hydrogel since any residual glutaraldehyde would have been toxic to the cells. The mesh was placed on top of the polymer along with cells passaged from the second dish and 160 mL of DMEM. The dish was placed back in the incubator.

The mesh was inspected on Day 3 and had settled into the PVA. The PVA coating the mesh was removed and the mesh was placed in another circular polystyrene dish with 160 mL of DMEM. The mesh was seeded with additional cells and placed back in the incubator.

On Days 4 and 5, examination of the mesh showed that some cells had attached and had begun to fill in the pores of the mesh (Figures 17 and 18). The two figures below, we elected to show different areas of the mesh. The cells in Figure 18 remained in Figure 19 (Day 4 and Day 5), therefore figures simply display separate areas of the mesh. The media was then changed on Day 5.

Figure 18: Fibroblast cells growing in pores of Vicryl mesh on Day 4 after seeding (100x magnification)

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Figure 19: Fibroblast cells growing in pores of Vicryl mesh on Day 5 after seeding (100x magnification)

Examination of the mesh on Day 7 showed continued growth of the cells on the mesh with cells beginning to span some of the pores as shown in Figure 20. Media was also changed on Day 7.

Figure 20: Fibroblast cells growing in pores of Vicryl mesh on Day 7 after seeding (100x magnification)

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At some point between Day 7 and Day 11, mold began to grow in the culture dish containing the mesh. Figure 21 shows a photograph of this extensive mold growth. Very few viable cells could be located on the mesh. The mesh was relocated to another polystyrene dish and additional antibiotic/antimycotic agent was added to the DMEM added to the new dish in an attempt to control the mold growth.

Figure 21: Mold and cells growing in pores of Vicryl mesh on Day 11 after seeding (100x magnification)

The attempt to control the mold growth was not successful, and on Day 13, the reactor and mesh were bleached and discarded.

Effects of Growth FactorsStatistical analysis using JUMP was done on the results from this experiment (see appendix B). There was no significant effect (within 5 %) from varying the concentrations of the growth factors.

DISCUSSIONExperimentationDue the results of our growth factor concentration experimentation, standard media was used. Testing the bioreactor protocol indicated that the design of the system promoted excellent mixing, as can be seen in the visible eddies in Figures 8-11. Furthermore, the recycle inlet stream created the desired level of circulation and turbulent mixing in the bioreactor. As predicted, the circular shape of the reactor prevented the creation of stagnant pools of media. Furthermore, adherence of healthy cells to the polymer scaffold was observed within 24 hours subsequent to the seeding of NIH/3T3 fibroblast cells.

However, due to complications and time restraint, the bioreactor was never used for our desired application, and therefore the keratinocytes were never seeded. Some of the

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problems and delays that occurred could have been avoided by either using a different method of seeding, or by using a smaller pore sized polymer. Also the multiple attempts at seeding increased exposure of our system, and therefore increased the risk of contamination. This could have been avoided by creating a different method of seeding such as using a rotating seeding bag; or by using a separate batch reactor for seeding purposes, and then transferring the construct to the bioreactor.

Actual productionThe protocol cell lines used were mouse cells, and the actual production would be done with human cells. The scale up of our construct could be done directly by increasing the size of the bioreactor and tubing, or by creating several bioreactors in parallel. Also, during production medium would need to be periodically tested for glucose levels in order to predict the precise time to harvest. In order to maintain extensive sterility and ease of handling, the reactor could be used as the shipping container for the construct. The system would need to be shipped and stored at approximately -70 °C. Another option would be to adhere the constructs into ethylvinylacetate (EVA) bags to prevent movement during growth or loss of implant during rinse previous to implantation.

There are several opportunities for future research in this area. Completion of the initial project proposal, including seeding of the keratinocytes, and placement of the construct in the continuous bioreactor should be attempted. Alternative applications of the product, such as pressure sores, cosmetic surgery, or diabetic ulcers could be investigated.

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REFERENCES

1. Online source. http://www.mccg.org/adulthealth/pmr/burn.asp, the Medical Center Online, The Medical Center of Central Georgia.

2. Halberstadt, C.R., Hardin, R., Bezverkov, K., Snyder, D., Allen, L., Landeen, L. The In Vitro Growth of a Three-Dimensional Human Dermal Replacement Using a Single-Pass Perfusion System. Biotechnol. Bioeng. 43(8): 740-746. 1994

3. Demling, R., DeSanti, L., Orgill, D., Online source. www.burnsurgery.org. 2000

4.Choudhary, N., Online source. Canadian Doctors Try Out New Artificial Skin. http://exn.ca/Stories/1996/12/24/01.asp. 2002

5. Online Source. How Organogenisis Makes Apligraft® www.organogenesis.com/howmade.htm. Organogenisis. 2002.

6. Goodwin, C.W., Maguire, M.S., McManus, W.F., and Pruitt, B.A., Jr. Prospective study of burn wound excision of dorsal burns of the hand. Plast Reconstr. Surg. 69: 670, 1982.

7.Online source. www.eng.buffalo.edu/Courses/ce435/2001ZGu/Polymers_in_medicine/ PolymersInMedicineReport.htm.

8. Cooper, M.L., Hansbrough, J.F., Spielvogel, R.L., Cohen, R., Bartel, R.L., Naughton, G.K.. In vivo optimization of a living dermal substitute employing cultured human fibroblasts on a biodegradable polyglycolic acid or polyglactin mesh. Biomaterials 12: 243-248. 1991

9. Freshney, R. I. Culture of Animal Cells: A Manual of Basic Technique. New York: Wiley-Liss.577p. 2000.

10. J.C., A.J., Tipton, Synthetic Biodegradable Polymers as Medical Devices. Medical Plastics and Biomaterials. Online source. http://www.devicelink.com/mpb/archive/98/03/002.html., 1999. 11. Figure reproduced courtesy of Journal of Biomedical Materials Research, 11:711, 1977.

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Appendix A

Fibroblast seeding density and media usage calculations

- For 3 in. by 3 in. Vicryl mesh (9 cm2 = 58 cm2)

- From Halberstadt et al., should get approximately 10 layers of cells on mesh- Total growth area = (58 cm2)(10 layers) = 580 cm2

- Halberstadt et al. used:- 24 in2 mesh seeded with 3.0 x 107 cells (6.0 x 105 cells/mL) with initial media

volume of 50 mL- Put in roller bottle for 24 h to facilitate seeding- Grew in static process for additional 24 h- Grew in single-pass perfusion system at 5.0-8.5 mL/h of media in 120 mL total

volume- Thus, media residence times: tau5.0 = 24 h and tau8.5 = 14 h- Grew for 16 days (384 h)- Thus, total media usage equal to ~2 L (125 mL/day)

- Apply to our system:

€

3.0x107 cells

24 in2 =1.13x107cells

9in2

2

5

2

5

9

1025.2

24

100.6

inmL

cellsx

inmL

cellsx

=

- Corning (http://www.corning.com/Lifesciences/technical_information/techDocs/Subculturing_protocol.pdf) recommends 0.2-0.3 mL of media per cm2 of growth area- Static media = (580 cm2)(0.25 mL/cm2) = 145 mL

- For 150 mm diameter dish (A = 176.7 cm2)- Maximum working height = 20 mm→ Volume = 354 mL

15 mm → Volume = 265 mL10 mm → Volume = 177 mL8.2 mm→ Volume = 145 mL

4 mm → Volume = 71 mL- Minimum working height = 3 mm → Volume = 53 mL (Just to top of one earring backing on post)

- Use ~160 mL for fibroblast growth and initial keratinocyte seeding- Use ~50-60 mL for keratinocyte air/liquid interface, could add additional earring backs to bring up level if need more media

- Media flow rates (with maximum recycle at all times)

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- With recycle, probably do not need tau < 24 h, but start with tau = 24 h for base case- Flow rate = 160 mL / 24 h = 7.0 mL/h

- Compare seeding with ATCC recommendations for NIH/3T3 fibroblasts(http://www.atcc.org/SearchCatalogs/longview.cfm?view=ce,1017912,CRL-1658&text=crl%2D1658&max=20)

- ATCC gives 102-105 cells for 100 mm diameter dish (A = 78.5 cm2)

€

105 cells

78.5cm2=1274

cells

cm2→(x580cm2)= 7.4 x105 cells

- Need to increase due to lower attachment efficiency (mesh efficiency is less than flat plate efficiency)

- Final seeding determination: 3 x 107 cells in 160 mL of media (1.9 x 105 cells/mL)

Keratinocyte seeding calculations- Do not seed until fibroblasts have achieved confluence on mesh

- If assume 3.0 x 107 gives approximately 10 layers of cells, then for 1 layer of keratinocytes:

€

3.0 x107 cells

10layers= 3.0x106 cells

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Appendix B

Growth Factor Experimental Data and Analysis

GROWTH FACTOR

Experiment ID Growth Factor concentration (ng/mL)

1 Control 02 bFGF (hi) 203 bFGF (low) 54 EGF (hi) 205 EGF (low) 56 bFGF/EGF (1:1) 10:107 bFGF/EGF (1:4) 4:168 bFGF/EGF (4:1) 16:4

t-Test: Paired Two Sample for Means

t-Test: Paired Two Sample for Means

Control bFGF (hi) ControlMean 25.91666667 20.08333333 Mean 25.91666667Variance 182.1458333 10.02083333 Variance 182.1458333Observations 3 3 Observations 3Pearson Correlation 0.66806357 Pearson Correlation -0.876389297Hypothesized Mean Difference

0 Hypothesized Mean Difference

0

df 2 df 2t Stat 0.869313726 t Stat -0.522553924P(T<=t) one-tail 0.238163569 P(T<=t) one-tail 0.326701254t Critical one-tail 2.91998731 t Critical one-tail 2.91998731P(T<=t) two-tail 0.476327138 P(T<=t) two-tail 0.653402509t Critical two-tail 4.302655725 t Critical two-tail 4.302655725

t-Test: Paired Two Sample for Means

t-Test: Paired Two Sample for Means

Control bFGF (low) ControlMean 25.91666667 25 Mean 25.91666667Variance 182.1458333 50.25 Variance 182.1458333Observations 3 3 Observations 3Pearson Correlation -0.665043636 Pearson Correlation -0.337897104Hypothesized Mean Difference

0 Hypothesized Mean Difference

0

df 2 df 2t Stat 0.083721028 t Stat -0.30655233P(T<=t) one-tail 0.470451879 P(T<=t) one-tail 0.394077323t Critical one-tail 2.91998731 t Critical one-tail 2.91998731P(T<=t) two-tail 0.940903758 P(T<=t) two-tail 0.788154646t Critical two-tail 4.302655725 t Critical two-tail 4.302655725

t-Test: Paired Two Sample for Means

t-Test: Paired Two Sample for Means

Control EGF (hi) Control

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Mean 25.91666667 24.25 Mean 25.91666667Variance 182.1458333 47.6875 Variance 182.1458333Observations 3 3 Observations 3Pearson Correlation -0.683013287 Pearson Correlation -0.28417169Hypothesized Mean Difference

0 Hypothesized Mean Difference

0

df 2 df 2t Stat 0.152751887 t Stat -0.037241946P(T<=t) one-tail 0.446306355 P(T<=t) one-tail 0.486837547t Critical one-tail 2.91998731 t Critical one-tail 2.91998731P(T<=t) two-tail 0.892612709 P(T<=t) two-tail 0.973675094t Critical two-tail 4.302655725 t Critical two-tail 4.302655725

t-Test: Paired Two Sample for Means

Control EGF (low)Mean 25.91666667 41.66666667Variance 182.1458333 10.58333333Observations 3 3Pearson Correlation -0.035113136Hypothesized Mean Difference

0

df 2t Stat -1.949489854P(T<=t) one-tail 0.095277017t Critical one-tail 2.91998731P(T<=t) two-tail 0.190554033t Critical two-tail 4.302655725

NOTE: In all instances, the calculated t value does NOT exceed the expected t value (for alpha=.05)Therefore, must FAIL TO REJECT a hypothesis of no difference between Control and each experimental case.

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Appendix C

Product Insert for PLGA Polymer

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