- Email: [email protected]
Human Tendon Cell Response to 7 Commercially Available Extracellular Matrix Materials: An In Vitro Study
Human Tendon Cell Response to 7 Commercially Available Extracellular Matrix Materials: An In Vitro Study Kevin P. Shea, M.D., Mary Beth McCarthy, B.S., Felicia Ledgard, B.S., Cristina Arciero, B.A., David Chowaniec, B.S., and Augustus D. Mazzocca, M.S., M.D.
Purpose: To evaluate the response of human tenocytes in culture to 7 commercially available extracellular matrix (ECM) patches. Methods: Four samples of each ECM were incubated in human tenocyte cultures by use of standard methods. Cell adhesion, cell proliferation, and cellular production of type I and type III collagen, decorin, and scleraxis were measured for each sample according to established experimental methods. Histologic samples were examined to measure the migration of the tenocytes into the ECM. Results: Tenocytes adhered more to samples of layered submucosal pig intestine than the 6 other ECM materials (P ⬍ .002). Tenocytes proliferated more and produced more matrix proteins when cultured on ECM derived from unaltered dermal specimens of human or porcine origin (P ⬍ .001). Cells were not seen to have migrated into the matrix of any ECM sample. Conclusions: Human tenocytes reacted most favorably to dermal ECM samples that were not chemically cross-linked by the manufacturer. Less favorable responses of the human cells were seen when cultured with equine or synthetic ECM, which showed favorable biologic responses in nonhuman models. Cellular migration into the matrix of the ECM is a complex process and cannot be replicated in this model entirely. Clinical Relevance: The results of this study suggest that dermal ECM may more favorably react with human tendon tissue than ECM of other origins. This may have great relevance as research continues in the field of augmenting surgical soft-tissue repair.
R
ecent studies have shown that surgically repaired rotator cuff tendons do not always heal.1-4 For many patients, this failure of healing is not symptomatic.1-4 However, for others, the pain and dysfunction from the torn rotator cuff continue, presenting the surgeon with a technical challenge: how to success-
From the Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, Connecticut, U.S.A. Recipient of the 2009 ISAKOS Scientific Award, ISAKOS Congress, Osaka, Japan, April 2009. Supported by Arthrex, Naples, Florida. K.P.S. is a consultant for Tornier, Edina, Minnesota. A.D.M. is a consultant for Arthrex. Received July 27, 2009; accepted January 22, 2010. Address correspondence and reprint requests to Kevin P. Shea, M.D., Department of Orthopaedic Surgery, University of Connecticut Health Center, Medical Arts and Research Building, 263 Farmington Ave, Farmington, CT 06034-4038, U.S.A. E-mail: [email protected] © 2010 by the Arthroscopy Association of North America 0749-8063/9441/$36.00 doi:10.1016/j.arthro.2010.01.020
fully repair a rotator cuff tear that has already failed to heal with the first surgery. Extracellular matrix (ECM) grafts have been used in some settings to mechanically reinforce the repair site.5 These patches are fashioned from a variety of biologic and engineered materials.5 The intent in using ECM materials is to provide mechanical support to the repair site, become incorporated into the host tissue, and then be replaced by the host tissue, completing the tendon-to-bone repair (S. Arnoczsky, D.V.M., oral personal communication, February 2008). ECM grafts have been studied extensively in animal models and show excellent incorporation into the host tissue. Human dermis,6-8 porcine dermis,9 fetal bovine dermis,8 pig small intestine submucosa (SIS),7-10 and synthetic materials made of woven polycarbonate polyurethane11 have been shown to produce satisfactory repairs of tendon tissue in canine,6 rat,7,8,11 and sheep9,10 models, without immunologic rejection. All of these studies conclude that the respective material may be well suited for human rotator cuff tendon repair augmentation.
Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 26, No 9 (September), 2010: pp 1181-1188
1181
1182
K. P. SHEA ET AL.
The findings of clinical reports using these ECM grafts in rotator cuff repair, however, have been very disappointing. Surgical failures, usually by graft rejection, have been reported with human rotator cuff tendon allograft,12 porcine dermis,13 SIS,14,15 synthetics,16 and equine pericardium.5 There are a few small studies that report favorable results with porcine dermis17 and freeze-dried human dermis.18 Thus there is a dichotomy between the laboratory studies and the clinical outcomes when ECM grafts are used to augment human rotator cuff repair. Therefore it is possible that a human tissue reaction to ECM materials may be substantially different than other species’ responses and that this difference may explain the variation between the animal and human studies. The purpose of this study was to examine the reaction of human tenocytes in culture to 7 commercially available ECM materials. Our hypothesis was that there would be a significant difference in adhesion, proliferation, and gene expression in the human tenocyte reaction to the 7 ECM grafts. METHODS Cell Culture In our laboratory fresh human tendons (long head of the biceps, semitendinosus, and gracilis), discarded from orthopaedic procedures, were immediately obtained and processed. For consistency, samples were obtained from healthy men, aged 18 to 60 years. The tissue was cut into small pieces and digested with 2 mg/mL of collagenase for 3.5 hours at 37°C, pelleted, resuspended, and plated into 100-mm (10-cm2) Falcon Primaria culture dishes (BD Biosciences, San Jose, CA) containing Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, 100 U/mL of penicillin, and 100 g/mL of streptomycin. Samples of each tendon isolation and subsequent cultures were routinely analyzed for protein and gene expression by use of Western blot and quantitative polymerase chain reaction (PCR) for tendon markers (decorin, types I and III collagen, and scleraxis). The cells were fed twice daily and grown to confluence, trypsinized, and plated onto each biomaterial specimen at a concentration of 40,000 cells/mL.19 For all experiments, tissue culture plastic (TCP) was used as the control and only passage 1 cells were used. ECM Patches All nonhuman patches are classified by the Food and Drug Administration as implant devices. GraftJacket
(Wright Medical Technology, Arlington, TX) is derived from freeze-dried human dermis cleansed in a proprietary process. It is not sterilized or chemically crosslinked. It is regulated as human allograft tissue. Conexa (Tornier, Edina, MN) is derived from porcine dermis. It is sterilized by a proprietary process and further prepared by removal of ␣-galactose residues to minimize human immunologic reaction to nonprimate, mammalian tissue. It is not chemically crosslinked. Collagen Repair Patch (Zimmer, Warsaw, IN) is derived from porcine dermis and sterilized by a proprietary process, and the collagen is chemically cross-linked to increase strength. Human fascia lata (Musculoskeletal Transplant Foundation, Edison, NJ) is human tissue, harvested through a sterile technique and fresh frozen. It is not commercially altered after harvest. OrthADAPT (Pegasus Biologics, Irvine, CA) is derived from equine pericardium. It is sterilized by a proprietary process and chemically cross-linked. Restore (SIS) (DePuy Orthopaedics, Warsaw, IN) is a preparation of 10 layers of submucosal porcine intestine. It is sterilized by a proprietary process and is not cross-linked. SportMesh (Biomet Sports Medicine, Warsaw, IN) is a biodegradable woven mesh composed of polyurethane urea that is provided in sterile condition. These 7 commercially available ECM materials were obtained directly from their manufacturers and are summarized in Table 1. All nonhuman ECM devices are approved by the Food and Drug Administration for reinforcement of tissue repairs but are not approved for the purpose of bridging tissue gaps (www .clinicaltrials.gov). Experimental Rationale Each of the ECM devices was examined by 4 experimental methods: cell adhesion, cell proliferation, cell activity, and histology. Sample size was limited to n ⫽ 4 per assay because of a limited supply of ECM materials. Although a larger sample size would have been ideal, n ⫽ 4 was believed to be sufficient to study the 4 different parameters (cell adhesion, proliferation, differentiation, and migration) assessed in this article. To accomplish a successful integration of the ECM into the host tissue, the tenocytes must attach to the ECM, proliferate, produce matrix, and ultimately, migrate into the ECM. Each of these studies examines 1 important aspect of the host’s reaction to an ECM graft. In the final analysis there is not agreement on which of these values is most important to successful graft-host integration. Instead, the determination of a favorable response by the host tissue to the ECM is a
HUMAN TENDON CELL RESPONSE TO ECM MATERIALS TABLE 1.
1183
Trade Name, Manufacturer, Graft Source, Sterilization, and Cross-Linking of 7 Commercially Available ECM Patches Used for Incubation of Human Tenocyte Cultures
GraftJacket Conexa Collagen Repair Patch Fascia lata OrthADAPT Restore SportMesh
Manufacturer
Source
Sterilized
Cross-Linked
Wright Medical Technology Tornier Zimmer Musculoskeletal Transplant Foundation Pegasus Biologics DePuy Orthopaedics Biomet Sports Medicine
Human epidermis Porcine epidermis Porcine dermis Human iliotibial band Equine pericardium Porcine intestinal submucosa Knitted Artelon
N Y Y N Y Y Y
N N Y N Y N N
summation of all experimental values. Cell adhesion measures the affinity of the host tissue to the ECM: the higher the value, the more favorable the response. Cell proliferation measures the tenocytes’ reaction to the ECM: the more proliferation, the more favorable the response. Cellular activity is measured by use of quantitative PCR, which measures the cellular production of messenger RNA (mRNA) that ultimately will be transcribed into proteins. Type I collagen and type III collagen are important tendon matrix structural proteins. Scleraxis is an important transcription factor in tendon growth and healing. Decorin is a predominant proteoglycan found in tendon ECM. The higher the values, the more favorable the response. Histologic analysis determines whether the tenocytes migrated into the ECM. Cell Adhesion Assay Four 5 ⫻ 5–mm samples of each of the 7 ECM devices were placed into tissue culture plates (BD Biosciences), and human tenocytes were plated onto each sample at a concentration of 40,000 cells/cm2. Samples were incubated for 24 hours and removed from the well. The adherent cells were removed from the sample by adding 0.5 mL of trypsin and incubating the sample for 20 minutes at 37°C. The cells from each sample were diluted in 9.5 mL of normal saline solution and counted 3 times each in a Coulter counter (Coulter Electronics, Hialeah, FL), which was repeated 3 times for accuracy. Cell Proliferation Assay Four samples of each ECM were plated as described previously. At 24 hours, 5 Ci/mL of tritium-labeled ([3H]) thymidine (NEN, Boston, MA) was added to tag nuclear DNA. At 48 hours, the samples were removed and washed twice for 5 minutes with 10% trichloroacetic acid to remove insoluble [3H]-thymidine. Cells were lysed for 10 minutes in 0.5 N sodium
hydroxide. Radioactivity was measured in the lysates with a liquid scintillation counter (Packard Instrument, Downers Grove, IL) and repeated 3 times for consistency. Cell Activity Four samples of each ECM were plated as described previously, harvested at 21 days, and moved to clean wells. All reactions were run in triplicate for accuracy. RNA was extracted by use of TRIzol reagent (Invitrogen [Gibco], Carlsbad, CA) according to the manufacturer’s recommendations. One microgram of total RNA was treated with DNase and reverse transcribed by use of the Reverse Transcriptase II kit and oligo-dT (Invitrogen [Gibco]). Reverse transcriptase–negative samples were prepared for each individual reaction and were used as controls for detection of assay contamination. The complementary DNA was used in 20-L PCRs containing 10 mmol/L of each specific primer (Applied Biosystems, Foster City, CA). The PCR was performed for 40 cycles of a 4-step program: 94°C for 30 seconds, annealing temperature for 20 seconds, 72°C for 20 seconds, and a fluorescence-read step for 20 seconds. The threshold cycles were obtained by use of Applied Biosystems software and were averaged. Glyceraldehyde 3-phosphate dehydrogenase served as an endogenous control. Histology Four samples of each ECM were incubated as described previously for 21 days. Specimens were harvested, dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in methyl methacrylate. Five-micrometer-thick cross sections of each sample were obtained, placed on a slide, and stained with H&E. Slides were examined qualitatively by a professional histomorphometrist (F.L.) using an OsteoMeasure computerized image analysis system (OsteoMetrics, Atlanta, GA) interfaced with an Op-
1184
K. P. SHEA ET AL.
FIGURE 1. Summary of cell adhesion to 7 ECM samples after 24 hours of incubation. Restore and GraftJacket showed significantly greater adhesion after 24 hours compared with any of the other samples tested (*P ⬍ .002 and P ⬍ .001, respectively). Cell adhesion to all of the other ECMs was not statistically different. TCP was used as a positive control. Bars represent the standard error of the mean.
tiphot Nikon microscope (Nikon, Melville, NY) at a magnification of ⫻20 to determine whether tenocytes had migrated into the ECM. Statistical Analysis Statistical analysis was performed by 1-way analysis of variance followed by the Student t test to determine significance between groups at a level of significance of P ⬍ .05. A post hoc Bonferroni adjustment was performed to confirm the results of multiple comparisons at a level of significance of P ⬍ .05. RESULTS Cell Adhesion The results of the cell adhesion assay are shown in Fig 1 and are expressed as the number of cells that adhered to the sample in 24 hours.20 Tenocytes ad-
FIGURE 2. Cell proliferation on surface of 7 ECM samples. Tenocytes proliferated equally on the 3 dermal ECMs (GraftJacket, Conexa, and Collagen Repair Patch) (P ⬎ .08). Tenocytes proliferated more on these 3 ECMs than on the remaining 4 ECMs (P ⬍ .001). TCP was used as a control. Bars represent the standard error of the mean. DPM refers to disintegrations per minute of the radioactive thymidine used in this assay. Asterisks indicate results are not statistically different from other ECM data bars with asterisks and are significantly greater than bars without asterisks.
hered more to the Restore sample than to any other ECM (P ⬍ .002). Cells adhered to GraftJacket more than any other device except Restore (P ⬍ .001). Cell adhesion to all of the other ECM materials was not statistically different. TCP was used as a positive control. Cell Proliferation The results of the cell proliferation assay are shown in Fig 2, are expressed as radioactive disintegrations per minute, and describe the relative amounts of [3H]thymidine incorporated into cellular DNA as a measure of cell division and proliferation.20 Tenocytes proliferated equally on the 3 dermal ECMs (GraftJacket, Conexa, and Collagen Repair Patch) (P ⬎ .08). Tenocytes proliferated more on these 3 ECMs than on the remaining 4 ECMs (P ⬍ .001). TCP was again used as a positive control.
HUMAN TENDON CELL RESPONSE TO ECM MATERIALS Cell Activity The results of the assays for cellular production of mRNA directed at the transcription of type I collagen, type III collagen, scleraxis, and decorin are shown graphically in Fig 3. Results are expressed as a percentage of control.21,22 Cells cultured on Conexa produced more mRNA for all 4 matrix components than they did on any of the other materials with the exception of type I collagen, where SportMesh was equivalent to Conexa, and scleraxis, where GraftJacket and SportMesh were equivalent to Conexa. Data for Collagen Repair Patch were similar to GraftJacket for type III collagen and decorin but lower for type I collagen and scleraxis. Values for OrthADAPT were similar to GraftJacket for decorin only and lower for the other 3 matrix components. Values for fascia lata and Restore were consistently lower than those for GraftJacket and Conexa for all 4 assays. Values for SportMesh were similar to those for GraftJacket and Conexa for type I collagen and scleraxis but lower for type III collagen and decorin. All comparisons were statistically significant at P ⱕ .001 or greater. Histology Representative H&E-stained sections of samples are shown in Fig 4. To observe positive or negative cell migration into the samples, observations were made in a blinded, nonbiased manner by 3 independent observers ( ⫽ 0.82). Cells were seen on the border of all devices, but no cells were seen in the internal matrix of any of the samples.
DISCUSSION The purpose of this study was to examine the response of human tenocytes to 7 available ECM grafts. The intent in using ECM in tendon repair is to mechanically protect the repair site and then for the ECM to become incorporated into the host tissue. It stands to reason that the specific human tendon cell response is very important to the incorporation of the ECM device. Although in vivo data exist on each individual scaffold material, these studies were performed in nonprimate animals that may have different responses than humans to the various ECM materials. In addition, several of these devices are derived from a variety of animal sources, and species-specific reactions to xenograft material can vary considerably. We believe that this is the first study to examine the response of human cells to implanted ECM materials.
1185
Our interpretation of the results is that Conexa and secondarily GraftJacket evoked the most favorable responses from the human tenocytes. Although cellular adhesion was lower for Conexa than for several other devices, the cells that adhered to both Conexa and GraftJacket, both composed of dermal tissue, showed more cellular proliferation and more production of mRNA directed toward transcription of all 4 matrix components than any of the other ECMs studied. Whereas cellular proliferation was also high in the Collagen Repair Patch, production of mRNA for type I and type III collagen and scleraxis was low. Cellular adhesion was the greatest with the Restore device, but the cells that did adhere to the material exhibited low cellular activity in every assay. Cellular adhesion, proliferation, and matrix production responses were mixed in the remaining 3 materials. In evaluating the data, it is important to keep in mind that the importance of high values in 1 assay versus another (e.g., mRNA directed at type I collagen v decorin) is not fully understood. Instead, we believed that high values in all assays, especially in cellular activity after cell adhesion, were most representative of a favorable host response to the ECM devices. Alterations in the collagen matrix appeared to have an effect on the tenocyte response as well. More favorable responses were seen for Conexa and GraftJacket, which are not subjected to chemical cross-linking of the matrix collagen, than for Collagen Repair Patch and OrthADAPT, which have the collagen chemically cross-linked during processing to increase material strength. Fascia lata is dense collagen with little matrix and had a similarly less favorable response. The matrix of the Restore device is a highly altered assembly of 10 layers of SIS and also evoked a less favorable tenocyte response. The results of this study are in agreement with the few published clinical reports of ECM use in human rotator cuff repair. Burkhead et al.18 reported favorable results in 12 of 17 patients with massive rotator cuff tears treated with open repair and augmented with GraftJacket. No infections or graft rejections were noted. Badhe et al.17 reported intact ultrasound-proven rotator cuff tendons in 8 of 10 patients with extensive rotator cuff tears surgically repaired and augmented with Rotator Collagen Repair Patch (Zimmer) at a follow-up averaging 4.5 years. However, Soler et al.13 reported failure in all 4 rotator cuff repairs done with Collagen Repair Patch at 3 to 6 months after implantation. No graft rejection response was reported in either study. Iannotti et al.15 and Walton et al.14 both reported high rates of failure and severe inflammatory
1186
K. P. SHEA ET AL.
FIGURE 3. Measure of mRNA directed toward type I collagen, type III collagen, decorin, and scleraxis produced by human tenocytes cultured on 1 of 7 ECM samples. Both Conexa and GraftJacket expressed significantly higher levels of mRNA for all tendon markers than the majority of other ECM samples. Fascia lata, when compared with the other ECM samples, expressed significantly lower mRNA levels. Comparisons between all ECM samples were statistically significant (*P ⱕ .001). Bars represent the standard error of the mean.
HUMAN TENDON CELL RESPONSE TO ECM MATERIALS
Fascia Lata ®
FIGURE 4. Human tenocytes are seen on the surface of these representative ECM samples (arrows). No migration of tenocytes into the matrix of any of the 7 ECM samples was observed after 21 days in culture. (H&E stain, original magnification ⫻20.)
Restore ®
Conexa ®
Collagen Repair Patch®
SportMesh®
OrthoAdapt®
GraftJacket®
responses when using the Restore device to augment rotator cuff repairs and discouraged its use. There are no published clinical reports on the outcomes using
1187
CuffPatch®
any of the other devices. In summary, the few published clinical studies show more favorable outcomes when using un– cross-linked dermal grafts than either
1188
K. P. SHEA ET AL.
cross-linked dermal grafts or submucosal porcine grafts. This trend is supported by our study. We did not see any cellular infiltration of any of the devices by the tenocytes. We removed the devices from the cell culture at 21 days because the cells begin to show altered morphology and diverse behavior after this time point, and a consistent phenotypic could not be relied upon thereafter. It remains unknown whether the cells would have infiltrated with more time. Fini et al.23 similarly were not able to show infiltration of porcine fibroblasts into a biologic matrix in a cell culture model. It is likely that cellular infiltration involves more than 1 cell line, a process that we are not able to replicate in this model. The results of this study are very preliminary because there are many limitations to our model. The process of host incorporation of graft materials is highly complex and cannot be replicated in any 1 model. The effect of the mechanical environment, surgical technique, and host tendon degeneration may also play a role in the success of any ECM-augmented surgical tendon repair. In our human tenocyte model, unaltered porcine and human dermal grafts elicited a more favorable response than did the other ECM devices, and these results support the results of the few clinical studies that have been published.
CONCLUSIONS Human tenocytes reacted most favorably to dermal ECM samples that were not chemically cross-linked by the manufacturer. Less favorable responses of the human cells were seen when cultured with equine or synthetic ECM devices, which showed favorable biologic responses in nonhuman models. Cellular migration into the matrix of the ECM is a complex process and cannot be replicated in this model entirely. REFERENCES 1. Harryman DT II, Mack LA, Wang KY, et al. Repairs of the rotator cuff: Correlation of functional results with integrity of the cuff. J Bone Joint Surg Am 1991;73:982-989. 2. Gazielly DF, Gleyze P, Montagnon C. Functional and anatomical results after rotator cuff repair. Clin Orthop Relat Res 1994:43-53. 3. Galatz LM, Ball CM, Teefet SA, Middleton WD, Yamagucci K. The outcome and repair integrity of completely arthroscopically repaired large and massive rotator cuff tears. J Bone Joint Surg Am 2004;86:219-224.
4. Fealey S, Adler RS, Drakos MC, et al. Patterns of vascular and anatomical response after rotator cuff repair. Am J Sports Med 2006;34:120-127. 5. Coons DA, Barber FA. Tendon graft substitutes—Rotator cuff patches. Sports Med Arthrosc Rev 2006;14:185-190. 6. Adams JE, Zobitz ME, Reacr JS Jr, An K-N, Steinmann SP. Rotator cuff repair using an acellular dermal matrix graft. An in vivo study in a canine model. Arthroscopy 2006;22:700-709. 7. Fina M, Torricelli P, Giavaresi G, et al. In vitro study comparing two collagenous membranes in view of their clinical application for rotator cuff tendon regeneration. J Orthop Res 2007;25:98-107. 8. Valentin JE, Badylak JS, McCabe GP, Badylak SF. Extracellular matrix bioscaffolds for orthopaedic applications. J Bone Joint Surg Am 2006;88:2673-2686. 9. Nicholson GP, Breur GJ, Van Sickle D, et al. Evaluation of a cross-linked acellular porcine dermal patch for rotator cuff repair augmentation in an ovine model. J Shoulder Elbow Surg 2007;16:S184-S191 (Suppl). 10. Schlegel TF, Hawkins RJ, Lewis CW, et al. The effects of augmentation with swine small intestine submucosa on tendon healing under tension: Histologic and mechanical evaluations in sheep. Am J Sports Med 2006;34:275-280. 11. Cole BJ, Gommoll AH, Yanke A, et al. Biocompatibility of a polymer patch for rotator cuff repair. Knee Surg Sports Traumatol Arthrosc 2007;15:632-637. 12. Neviaser JS, Neviaser RJ, Neviaser TJ. The repair of chronic massive ruptures of the rotator cuff of the shoulder by use of freeze-dried rotator cuff. J Bone Joint Surg Am 1978;60:681684. 13. Soler JA, Gidwani S, Curtis MJ. Early complications from the use of porcine dermal collagen implants (Permacol) as bridging constructs in the repair of massive rotator cuff tears. A report of 4 cases. Acta Orthop Belg 2007;73:432-436. 14. Walton JR, Bowman NK, Khatib Y, et al. Restore Orthobiologic implant: Not recommended for augmentation of rotator cuff repairs. J Bone Joint Surg Am 2007;89:786-791. 15. Iannotti JP, Codsi MJ, Kwon YW, et al. Porcine small intestine submucosa augmentation of surgical repair of chronic twotendon cuff tears. J Bone Joint Surg Am 2006;88:1238-1244. 16. Audenaert E, VanNuffel J, Schepens A, et al. Reconstruction of massive rotator cuff lesions with a synthetic interposition graft: A prospective study of 41 patients. Knee Surg Sports Traumatol Arthrosc 2006;14:360-364. 17. Badhe SP, Lawrence TM, Smith FD, Lunn PG. An assessment of porcine dermal xenograft as an augmentation graft in the treatment of extensive rotator cuff tears. J Shoulder Elbow Surg 2008;17:S35-S39 (Suppl). 18. Burkhead WZ Jr, Schiffern SC, Krishnan SG. Use of graft jacket as an augmentation for massive rotator cuff tears. Semin Arthroplasty 2007;18:11-18. 19. Ashihara T, Baserga R. Cell synchronization. Methods Enzymol 1979;58:248-262. 20. Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11-25. 21. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159. 22. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986;51:263-273 (Pt 1). 23. Fini M, Bondioli ED, Torricelli P, et al. Decellularized human derma as a scaffold for the regeneration of rotator cuff tendons: In vitro investigations. Presented at the 21st Congress of the European Society for Surgery of the Shoulder and the Elbow, Bruges, Belgium, September, 2008.
Purpose: To evaluate the response of human tenocytes in culture to 7 commercially available extracellular matrix (ECM) patches. Methods: Four samples of each ECM were incubated in human tenocyte cultures by use of standard methods. Cell adhesion, cell proliferation, and cellular production of type I and type III collagen, decorin, and scleraxis were measured for each sample according to established experimental methods. Histologic samples were examined to measure the migration of the tenocytes into the ECM. Results: Tenocytes adhered more to samples of layered submucosal pig intestine than the 6 other ECM materials (P ⬍ .002). Tenocytes proliferated more and produced more matrix proteins when cultured on ECM derived from unaltered dermal specimens of human or porcine origin (P ⬍ .001). Cells were not seen to have migrated into the matrix of any ECM sample. Conclusions: Human tenocytes reacted most favorably to dermal ECM samples that were not chemically cross-linked by the manufacturer. Less favorable responses of the human cells were seen when cultured with equine or synthetic ECM, which showed favorable biologic responses in nonhuman models. Cellular migration into the matrix of the ECM is a complex process and cannot be replicated in this model entirely. Clinical Relevance: The results of this study suggest that dermal ECM may more favorably react with human tendon tissue than ECM of other origins. This may have great relevance as research continues in the field of augmenting surgical soft-tissue repair.
R
ecent studies have shown that surgically repaired rotator cuff tendons do not always heal.1-4 For many patients, this failure of healing is not symptomatic.1-4 However, for others, the pain and dysfunction from the torn rotator cuff continue, presenting the surgeon with a technical challenge: how to success-
From the Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, Connecticut, U.S.A. Recipient of the 2009 ISAKOS Scientific Award, ISAKOS Congress, Osaka, Japan, April 2009. Supported by Arthrex, Naples, Florida. K.P.S. is a consultant for Tornier, Edina, Minnesota. A.D.M. is a consultant for Arthrex. Received July 27, 2009; accepted January 22, 2010. Address correspondence and reprint requests to Kevin P. Shea, M.D., Department of Orthopaedic Surgery, University of Connecticut Health Center, Medical Arts and Research Building, 263 Farmington Ave, Farmington, CT 06034-4038, U.S.A. E-mail: [email protected] © 2010 by the Arthroscopy Association of North America 0749-8063/9441/$36.00 doi:10.1016/j.arthro.2010.01.020
fully repair a rotator cuff tear that has already failed to heal with the first surgery. Extracellular matrix (ECM) grafts have been used in some settings to mechanically reinforce the repair site.5 These patches are fashioned from a variety of biologic and engineered materials.5 The intent in using ECM materials is to provide mechanical support to the repair site, become incorporated into the host tissue, and then be replaced by the host tissue, completing the tendon-to-bone repair (S. Arnoczsky, D.V.M., oral personal communication, February 2008). ECM grafts have been studied extensively in animal models and show excellent incorporation into the host tissue. Human dermis,6-8 porcine dermis,9 fetal bovine dermis,8 pig small intestine submucosa (SIS),7-10 and synthetic materials made of woven polycarbonate polyurethane11 have been shown to produce satisfactory repairs of tendon tissue in canine,6 rat,7,8,11 and sheep9,10 models, without immunologic rejection. All of these studies conclude that the respective material may be well suited for human rotator cuff tendon repair augmentation.
Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 26, No 9 (September), 2010: pp 1181-1188
1181
1182
K. P. SHEA ET AL.
The findings of clinical reports using these ECM grafts in rotator cuff repair, however, have been very disappointing. Surgical failures, usually by graft rejection, have been reported with human rotator cuff tendon allograft,12 porcine dermis,13 SIS,14,15 synthetics,16 and equine pericardium.5 There are a few small studies that report favorable results with porcine dermis17 and freeze-dried human dermis.18 Thus there is a dichotomy between the laboratory studies and the clinical outcomes when ECM grafts are used to augment human rotator cuff repair. Therefore it is possible that a human tissue reaction to ECM materials may be substantially different than other species’ responses and that this difference may explain the variation between the animal and human studies. The purpose of this study was to examine the reaction of human tenocytes in culture to 7 commercially available ECM materials. Our hypothesis was that there would be a significant difference in adhesion, proliferation, and gene expression in the human tenocyte reaction to the 7 ECM grafts. METHODS Cell Culture In our laboratory fresh human tendons (long head of the biceps, semitendinosus, and gracilis), discarded from orthopaedic procedures, were immediately obtained and processed. For consistency, samples were obtained from healthy men, aged 18 to 60 years. The tissue was cut into small pieces and digested with 2 mg/mL of collagenase for 3.5 hours at 37°C, pelleted, resuspended, and plated into 100-mm (10-cm2) Falcon Primaria culture dishes (BD Biosciences, San Jose, CA) containing Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, 100 U/mL of penicillin, and 100 g/mL of streptomycin. Samples of each tendon isolation and subsequent cultures were routinely analyzed for protein and gene expression by use of Western blot and quantitative polymerase chain reaction (PCR) for tendon markers (decorin, types I and III collagen, and scleraxis). The cells were fed twice daily and grown to confluence, trypsinized, and plated onto each biomaterial specimen at a concentration of 40,000 cells/mL.19 For all experiments, tissue culture plastic (TCP) was used as the control and only passage 1 cells were used. ECM Patches All nonhuman patches are classified by the Food and Drug Administration as implant devices. GraftJacket
(Wright Medical Technology, Arlington, TX) is derived from freeze-dried human dermis cleansed in a proprietary process. It is not sterilized or chemically crosslinked. It is regulated as human allograft tissue. Conexa (Tornier, Edina, MN) is derived from porcine dermis. It is sterilized by a proprietary process and further prepared by removal of ␣-galactose residues to minimize human immunologic reaction to nonprimate, mammalian tissue. It is not chemically crosslinked. Collagen Repair Patch (Zimmer, Warsaw, IN) is derived from porcine dermis and sterilized by a proprietary process, and the collagen is chemically cross-linked to increase strength. Human fascia lata (Musculoskeletal Transplant Foundation, Edison, NJ) is human tissue, harvested through a sterile technique and fresh frozen. It is not commercially altered after harvest. OrthADAPT (Pegasus Biologics, Irvine, CA) is derived from equine pericardium. It is sterilized by a proprietary process and chemically cross-linked. Restore (SIS) (DePuy Orthopaedics, Warsaw, IN) is a preparation of 10 layers of submucosal porcine intestine. It is sterilized by a proprietary process and is not cross-linked. SportMesh (Biomet Sports Medicine, Warsaw, IN) is a biodegradable woven mesh composed of polyurethane urea that is provided in sterile condition. These 7 commercially available ECM materials were obtained directly from their manufacturers and are summarized in Table 1. All nonhuman ECM devices are approved by the Food and Drug Administration for reinforcement of tissue repairs but are not approved for the purpose of bridging tissue gaps (www .clinicaltrials.gov). Experimental Rationale Each of the ECM devices was examined by 4 experimental methods: cell adhesion, cell proliferation, cell activity, and histology. Sample size was limited to n ⫽ 4 per assay because of a limited supply of ECM materials. Although a larger sample size would have been ideal, n ⫽ 4 was believed to be sufficient to study the 4 different parameters (cell adhesion, proliferation, differentiation, and migration) assessed in this article. To accomplish a successful integration of the ECM into the host tissue, the tenocytes must attach to the ECM, proliferate, produce matrix, and ultimately, migrate into the ECM. Each of these studies examines 1 important aspect of the host’s reaction to an ECM graft. In the final analysis there is not agreement on which of these values is most important to successful graft-host integration. Instead, the determination of a favorable response by the host tissue to the ECM is a
HUMAN TENDON CELL RESPONSE TO ECM MATERIALS TABLE 1.
1183
Trade Name, Manufacturer, Graft Source, Sterilization, and Cross-Linking of 7 Commercially Available ECM Patches Used for Incubation of Human Tenocyte Cultures
GraftJacket Conexa Collagen Repair Patch Fascia lata OrthADAPT Restore SportMesh
Manufacturer
Source
Sterilized
Cross-Linked
Wright Medical Technology Tornier Zimmer Musculoskeletal Transplant Foundation Pegasus Biologics DePuy Orthopaedics Biomet Sports Medicine
Human epidermis Porcine epidermis Porcine dermis Human iliotibial band Equine pericardium Porcine intestinal submucosa Knitted Artelon
N Y Y N Y Y Y
N N Y N Y N N
summation of all experimental values. Cell adhesion measures the affinity of the host tissue to the ECM: the higher the value, the more favorable the response. Cell proliferation measures the tenocytes’ reaction to the ECM: the more proliferation, the more favorable the response. Cellular activity is measured by use of quantitative PCR, which measures the cellular production of messenger RNA (mRNA) that ultimately will be transcribed into proteins. Type I collagen and type III collagen are important tendon matrix structural proteins. Scleraxis is an important transcription factor in tendon growth and healing. Decorin is a predominant proteoglycan found in tendon ECM. The higher the values, the more favorable the response. Histologic analysis determines whether the tenocytes migrated into the ECM. Cell Adhesion Assay Four 5 ⫻ 5–mm samples of each of the 7 ECM devices were placed into tissue culture plates (BD Biosciences), and human tenocytes were plated onto each sample at a concentration of 40,000 cells/cm2. Samples were incubated for 24 hours and removed from the well. The adherent cells were removed from the sample by adding 0.5 mL of trypsin and incubating the sample for 20 minutes at 37°C. The cells from each sample were diluted in 9.5 mL of normal saline solution and counted 3 times each in a Coulter counter (Coulter Electronics, Hialeah, FL), which was repeated 3 times for accuracy. Cell Proliferation Assay Four samples of each ECM were plated as described previously. At 24 hours, 5 Ci/mL of tritium-labeled ([3H]) thymidine (NEN, Boston, MA) was added to tag nuclear DNA. At 48 hours, the samples were removed and washed twice for 5 minutes with 10% trichloroacetic acid to remove insoluble [3H]-thymidine. Cells were lysed for 10 minutes in 0.5 N sodium
hydroxide. Radioactivity was measured in the lysates with a liquid scintillation counter (Packard Instrument, Downers Grove, IL) and repeated 3 times for consistency. Cell Activity Four samples of each ECM were plated as described previously, harvested at 21 days, and moved to clean wells. All reactions were run in triplicate for accuracy. RNA was extracted by use of TRIzol reagent (Invitrogen [Gibco], Carlsbad, CA) according to the manufacturer’s recommendations. One microgram of total RNA was treated with DNase and reverse transcribed by use of the Reverse Transcriptase II kit and oligo-dT (Invitrogen [Gibco]). Reverse transcriptase–negative samples were prepared for each individual reaction and were used as controls for detection of assay contamination. The complementary DNA was used in 20-L PCRs containing 10 mmol/L of each specific primer (Applied Biosystems, Foster City, CA). The PCR was performed for 40 cycles of a 4-step program: 94°C for 30 seconds, annealing temperature for 20 seconds, 72°C for 20 seconds, and a fluorescence-read step for 20 seconds. The threshold cycles were obtained by use of Applied Biosystems software and were averaged. Glyceraldehyde 3-phosphate dehydrogenase served as an endogenous control. Histology Four samples of each ECM were incubated as described previously for 21 days. Specimens were harvested, dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in methyl methacrylate. Five-micrometer-thick cross sections of each sample were obtained, placed on a slide, and stained with H&E. Slides were examined qualitatively by a professional histomorphometrist (F.L.) using an OsteoMeasure computerized image analysis system (OsteoMetrics, Atlanta, GA) interfaced with an Op-
1184
K. P. SHEA ET AL.
FIGURE 1. Summary of cell adhesion to 7 ECM samples after 24 hours of incubation. Restore and GraftJacket showed significantly greater adhesion after 24 hours compared with any of the other samples tested (*P ⬍ .002 and P ⬍ .001, respectively). Cell adhesion to all of the other ECMs was not statistically different. TCP was used as a positive control. Bars represent the standard error of the mean.
tiphot Nikon microscope (Nikon, Melville, NY) at a magnification of ⫻20 to determine whether tenocytes had migrated into the ECM. Statistical Analysis Statistical analysis was performed by 1-way analysis of variance followed by the Student t test to determine significance between groups at a level of significance of P ⬍ .05. A post hoc Bonferroni adjustment was performed to confirm the results of multiple comparisons at a level of significance of P ⬍ .05. RESULTS Cell Adhesion The results of the cell adhesion assay are shown in Fig 1 and are expressed as the number of cells that adhered to the sample in 24 hours.20 Tenocytes ad-
FIGURE 2. Cell proliferation on surface of 7 ECM samples. Tenocytes proliferated equally on the 3 dermal ECMs (GraftJacket, Conexa, and Collagen Repair Patch) (P ⬎ .08). Tenocytes proliferated more on these 3 ECMs than on the remaining 4 ECMs (P ⬍ .001). TCP was used as a control. Bars represent the standard error of the mean. DPM refers to disintegrations per minute of the radioactive thymidine used in this assay. Asterisks indicate results are not statistically different from other ECM data bars with asterisks and are significantly greater than bars without asterisks.
hered more to the Restore sample than to any other ECM (P ⬍ .002). Cells adhered to GraftJacket more than any other device except Restore (P ⬍ .001). Cell adhesion to all of the other ECM materials was not statistically different. TCP was used as a positive control. Cell Proliferation The results of the cell proliferation assay are shown in Fig 2, are expressed as radioactive disintegrations per minute, and describe the relative amounts of [3H]thymidine incorporated into cellular DNA as a measure of cell division and proliferation.20 Tenocytes proliferated equally on the 3 dermal ECMs (GraftJacket, Conexa, and Collagen Repair Patch) (P ⬎ .08). Tenocytes proliferated more on these 3 ECMs than on the remaining 4 ECMs (P ⬍ .001). TCP was again used as a positive control.
HUMAN TENDON CELL RESPONSE TO ECM MATERIALS Cell Activity The results of the assays for cellular production of mRNA directed at the transcription of type I collagen, type III collagen, scleraxis, and decorin are shown graphically in Fig 3. Results are expressed as a percentage of control.21,22 Cells cultured on Conexa produced more mRNA for all 4 matrix components than they did on any of the other materials with the exception of type I collagen, where SportMesh was equivalent to Conexa, and scleraxis, where GraftJacket and SportMesh were equivalent to Conexa. Data for Collagen Repair Patch were similar to GraftJacket for type III collagen and decorin but lower for type I collagen and scleraxis. Values for OrthADAPT were similar to GraftJacket for decorin only and lower for the other 3 matrix components. Values for fascia lata and Restore were consistently lower than those for GraftJacket and Conexa for all 4 assays. Values for SportMesh were similar to those for GraftJacket and Conexa for type I collagen and scleraxis but lower for type III collagen and decorin. All comparisons were statistically significant at P ⱕ .001 or greater. Histology Representative H&E-stained sections of samples are shown in Fig 4. To observe positive or negative cell migration into the samples, observations were made in a blinded, nonbiased manner by 3 independent observers ( ⫽ 0.82). Cells were seen on the border of all devices, but no cells were seen in the internal matrix of any of the samples.
DISCUSSION The purpose of this study was to examine the response of human tenocytes to 7 available ECM grafts. The intent in using ECM in tendon repair is to mechanically protect the repair site and then for the ECM to become incorporated into the host tissue. It stands to reason that the specific human tendon cell response is very important to the incorporation of the ECM device. Although in vivo data exist on each individual scaffold material, these studies were performed in nonprimate animals that may have different responses than humans to the various ECM materials. In addition, several of these devices are derived from a variety of animal sources, and species-specific reactions to xenograft material can vary considerably. We believe that this is the first study to examine the response of human cells to implanted ECM materials.
1185
Our interpretation of the results is that Conexa and secondarily GraftJacket evoked the most favorable responses from the human tenocytes. Although cellular adhesion was lower for Conexa than for several other devices, the cells that adhered to both Conexa and GraftJacket, both composed of dermal tissue, showed more cellular proliferation and more production of mRNA directed toward transcription of all 4 matrix components than any of the other ECMs studied. Whereas cellular proliferation was also high in the Collagen Repair Patch, production of mRNA for type I and type III collagen and scleraxis was low. Cellular adhesion was the greatest with the Restore device, but the cells that did adhere to the material exhibited low cellular activity in every assay. Cellular adhesion, proliferation, and matrix production responses were mixed in the remaining 3 materials. In evaluating the data, it is important to keep in mind that the importance of high values in 1 assay versus another (e.g., mRNA directed at type I collagen v decorin) is not fully understood. Instead, we believed that high values in all assays, especially in cellular activity after cell adhesion, were most representative of a favorable host response to the ECM devices. Alterations in the collagen matrix appeared to have an effect on the tenocyte response as well. More favorable responses were seen for Conexa and GraftJacket, which are not subjected to chemical cross-linking of the matrix collagen, than for Collagen Repair Patch and OrthADAPT, which have the collagen chemically cross-linked during processing to increase material strength. Fascia lata is dense collagen with little matrix and had a similarly less favorable response. The matrix of the Restore device is a highly altered assembly of 10 layers of SIS and also evoked a less favorable tenocyte response. The results of this study are in agreement with the few published clinical reports of ECM use in human rotator cuff repair. Burkhead et al.18 reported favorable results in 12 of 17 patients with massive rotator cuff tears treated with open repair and augmented with GraftJacket. No infections or graft rejections were noted. Badhe et al.17 reported intact ultrasound-proven rotator cuff tendons in 8 of 10 patients with extensive rotator cuff tears surgically repaired and augmented with Rotator Collagen Repair Patch (Zimmer) at a follow-up averaging 4.5 years. However, Soler et al.13 reported failure in all 4 rotator cuff repairs done with Collagen Repair Patch at 3 to 6 months after implantation. No graft rejection response was reported in either study. Iannotti et al.15 and Walton et al.14 both reported high rates of failure and severe inflammatory
1186
K. P. SHEA ET AL.
FIGURE 3. Measure of mRNA directed toward type I collagen, type III collagen, decorin, and scleraxis produced by human tenocytes cultured on 1 of 7 ECM samples. Both Conexa and GraftJacket expressed significantly higher levels of mRNA for all tendon markers than the majority of other ECM samples. Fascia lata, when compared with the other ECM samples, expressed significantly lower mRNA levels. Comparisons between all ECM samples were statistically significant (*P ⱕ .001). Bars represent the standard error of the mean.
HUMAN TENDON CELL RESPONSE TO ECM MATERIALS
Fascia Lata ®
FIGURE 4. Human tenocytes are seen on the surface of these representative ECM samples (arrows). No migration of tenocytes into the matrix of any of the 7 ECM samples was observed after 21 days in culture. (H&E stain, original magnification ⫻20.)
Restore ®
Conexa ®
Collagen Repair Patch®
SportMesh®
OrthoAdapt®
GraftJacket®
responses when using the Restore device to augment rotator cuff repairs and discouraged its use. There are no published clinical reports on the outcomes using
1187
CuffPatch®
any of the other devices. In summary, the few published clinical studies show more favorable outcomes when using un– cross-linked dermal grafts than either
1188
K. P. SHEA ET AL.
cross-linked dermal grafts or submucosal porcine grafts. This trend is supported by our study. We did not see any cellular infiltration of any of the devices by the tenocytes. We removed the devices from the cell culture at 21 days because the cells begin to show altered morphology and diverse behavior after this time point, and a consistent phenotypic could not be relied upon thereafter. It remains unknown whether the cells would have infiltrated with more time. Fini et al.23 similarly were not able to show infiltration of porcine fibroblasts into a biologic matrix in a cell culture model. It is likely that cellular infiltration involves more than 1 cell line, a process that we are not able to replicate in this model. The results of this study are very preliminary because there are many limitations to our model. The process of host incorporation of graft materials is highly complex and cannot be replicated in any 1 model. The effect of the mechanical environment, surgical technique, and host tendon degeneration may also play a role in the success of any ECM-augmented surgical tendon repair. In our human tenocyte model, unaltered porcine and human dermal grafts elicited a more favorable response than did the other ECM devices, and these results support the results of the few clinical studies that have been published.
CONCLUSIONS Human tenocytes reacted most favorably to dermal ECM samples that were not chemically cross-linked by the manufacturer. Less favorable responses of the human cells were seen when cultured with equine or synthetic ECM devices, which showed favorable biologic responses in nonhuman models. Cellular migration into the matrix of the ECM is a complex process and cannot be replicated in this model entirely. REFERENCES 1. Harryman DT II, Mack LA, Wang KY, et al. Repairs of the rotator cuff: Correlation of functional results with integrity of the cuff. J Bone Joint Surg Am 1991;73:982-989. 2. Gazielly DF, Gleyze P, Montagnon C. Functional and anatomical results after rotator cuff repair. Clin Orthop Relat Res 1994:43-53. 3. Galatz LM, Ball CM, Teefet SA, Middleton WD, Yamagucci K. The outcome and repair integrity of completely arthroscopically repaired large and massive rotator cuff tears. J Bone Joint Surg Am 2004;86:219-224.
4. Fealey S, Adler RS, Drakos MC, et al. Patterns of vascular and anatomical response after rotator cuff repair. Am J Sports Med 2006;34:120-127. 5. Coons DA, Barber FA. Tendon graft substitutes—Rotator cuff patches. Sports Med Arthrosc Rev 2006;14:185-190. 6. Adams JE, Zobitz ME, Reacr JS Jr, An K-N, Steinmann SP. Rotator cuff repair using an acellular dermal matrix graft. An in vivo study in a canine model. Arthroscopy 2006;22:700-709. 7. Fina M, Torricelli P, Giavaresi G, et al. In vitro study comparing two collagenous membranes in view of their clinical application for rotator cuff tendon regeneration. J Orthop Res 2007;25:98-107. 8. Valentin JE, Badylak JS, McCabe GP, Badylak SF. Extracellular matrix bioscaffolds for orthopaedic applications. J Bone Joint Surg Am 2006;88:2673-2686. 9. Nicholson GP, Breur GJ, Van Sickle D, et al. Evaluation of a cross-linked acellular porcine dermal patch for rotator cuff repair augmentation in an ovine model. J Shoulder Elbow Surg 2007;16:S184-S191 (Suppl). 10. Schlegel TF, Hawkins RJ, Lewis CW, et al. The effects of augmentation with swine small intestine submucosa on tendon healing under tension: Histologic and mechanical evaluations in sheep. Am J Sports Med 2006;34:275-280. 11. Cole BJ, Gommoll AH, Yanke A, et al. Biocompatibility of a polymer patch for rotator cuff repair. Knee Surg Sports Traumatol Arthrosc 2007;15:632-637. 12. Neviaser JS, Neviaser RJ, Neviaser TJ. The repair of chronic massive ruptures of the rotator cuff of the shoulder by use of freeze-dried rotator cuff. J Bone Joint Surg Am 1978;60:681684. 13. Soler JA, Gidwani S, Curtis MJ. Early complications from the use of porcine dermal collagen implants (Permacol) as bridging constructs in the repair of massive rotator cuff tears. A report of 4 cases. Acta Orthop Belg 2007;73:432-436. 14. Walton JR, Bowman NK, Khatib Y, et al. Restore Orthobiologic implant: Not recommended for augmentation of rotator cuff repairs. J Bone Joint Surg Am 2007;89:786-791. 15. Iannotti JP, Codsi MJ, Kwon YW, et al. Porcine small intestine submucosa augmentation of surgical repair of chronic twotendon cuff tears. J Bone Joint Surg Am 2006;88:1238-1244. 16. Audenaert E, VanNuffel J, Schepens A, et al. Reconstruction of massive rotator cuff lesions with a synthetic interposition graft: A prospective study of 41 patients. Knee Surg Sports Traumatol Arthrosc 2006;14:360-364. 17. Badhe SP, Lawrence TM, Smith FD, Lunn PG. An assessment of porcine dermal xenograft as an augmentation graft in the treatment of extensive rotator cuff tears. J Shoulder Elbow Surg 2008;17:S35-S39 (Suppl). 18. Burkhead WZ Jr, Schiffern SC, Krishnan SG. Use of graft jacket as an augmentation for massive rotator cuff tears. Semin Arthroplasty 2007;18:11-18. 19. Ashihara T, Baserga R. Cell synchronization. Methods Enzymol 1979;58:248-262. 20. Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11-25. 21. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159. 22. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986;51:263-273 (Pt 1). 23. Fini M, Bondioli ED, Torricelli P, et al. Decellularized human derma as a scaffold for the regeneration of rotator cuff tendons: In vitro investigations. Presented at the 21st Congress of the European Society for Surgery of the Shoulder and the Elbow, Bruges, Belgium, September, 2008.