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ArticleTitle Adhesion and collagen production of human tenocytes seeded on degradable poly(urethane urea)Article Sub-Title

Article CopyRight Springer-Verlag Berlin Heidelberg(This will be the copyright line in the final PDF)

Journal Name Knee Surgery, Sports Traumatology, Arthroscopy

Corresponding Author Family Name RuzziniParticle

Given Name LauraSuffix

Division Department of Orthopaedic and Trauma Surgery, Center for IntegratedResearch

Organization Università Campus Bio-Medico

Address Via A. Del Portillo, 200, Rome, Italy

Division Department of Orthopedics

Organization Ospedale Pediatrico Bambino Gesù

Address Via Torre di Palidoro Palidoro, Rome, Italy

Email l.ruzzini@unicampus.it

Author Family Name LongoParticle

Given Name Umile GiuseppeSuffix

Division Department of Orthopaedic and Trauma Surgery, Center for IntegratedResearch

Organization Università Campus Bio-Medico

Address Via A. Del Portillo, 200, Rome, Italy

Email

Author Family Name CampiParticle

Given Name StefanoSuffix

Division Department of Orthopaedic and Trauma Surgery, Center for IntegratedResearch

Organization Università Campus Bio-Medico

Address Via A. Del Portillo, 200, Rome, Italy

Email

Author Family Name MaffulliParticle

Given Name NicolaSuffix

Division

Organization Centre for Sports and Exercise Medicine, Barts and The London School ofMedicine and Dentistry Mile End Hospital

Address 275 Bancroft Road, London, E1 4DG, UK

Email

Author Family Name MudaParticle

Given Name Andrea OnettiSuffix

Division Department of Pathology

Organization Università Campus Biomedico

Address Via A. Del Portillo, 200, Rome, Italy

Email

Author Family Name DenaroParticle

Given Name VincenzoSuffix

Division Department of Orthopaedic and Trauma Surgery, Center for IntegratedResearch

Organization Università Campus Bio-Medico

Address Via A. Del Portillo, 200, Rome, Italy

Email

Schedule

Received 28 June 2012

Revised

Accepted 9 October 2012

Abstract Purpose:The aim of this study was to investigate whether human tenocytes taken from ruptured quadriceps tendoncould be seeded on a biodegradable polycaprolactone-based polyurethanes (PU) urea scaffold. Scaffoldcolonization and collagen production after different culture periods were analyzed to understand whethertenocytes from ruptured tendons are able to colonize these biodegradable scaffolds.Methods:Human primary tenocyte cultures of ruptured quadriceps tendons were seeded on PU scaffolds. After 3, 10and 15 days of incubation, the samples were stained with haematoxylin and eosin and were examined underwhite light microscopy. After 15 and 30 days of incubation, samples were examined under transmissionelectron microscope. Total collagen accumulation was also evaluated after 15, 30 and 45 days of culture.Results:After 15 and 30 days of culture, tenocyte-seeded scaffolds showed cell colonization and cell accumulationaround interconnecting micropores. Tenocyte phenotype was variable. Collagen accumulation in seededscaffolds demonstrated a progressive increase after 15, 30 and 45 days of culture, while control non-seededscaffolds show no collagen accumulation.Conclusion:These results showed that human tenocytes from ruptured quadriceps tendon can be seeded onpolycaprolactone-based PU urea scaffolds and cultured for a long time period (45 days). This study alsoshowed that human tenocytes from ruptured tendons seeded on PU scaffolds are able to penetrate the scaffoldshowing a progressively higher collagen accumulation after 15, 30 and 45 days of incubation. This studyprovides the basis to use this PU biodegradable scaffold in vivo as an augmentation for chronic tendon rupturesand in vitro as a scaffold for tissue engineering construct.

Keywords (separated by '-') Tendon rupture - Human tenocytes - Tissue engineering - ScaffoldsFootnote Information

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Journal: 167

Article: 2249

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EXPERIMENTAL STUDY1

2 Adhesion and collagen production of human tenocytes seeded

3 on degradable poly(urethane urea)

4 Laura Ruzzini • Umile Giuseppe Longo •

5 Stefano Campi • Nicola Maffulli •

6 Andrea Onetti Muda • Vincenzo Denaro

7 Received: 28 June 2012 / Accepted: 9 October 20128 � Springer-Verlag Berlin Heidelberg 2012

9 Abstract

10 Purpose The aim of this study was to investigate whether

11 human tenocytes taken from ruptured quadriceps tendon

12 could be seeded on a biodegradable polycaprolactone-

13 based polyurethanes (PU) urea scaffold. Scaffold coloni-

14 zation and collagen production after different culture

15 periods were analyzed to understand whether tenocytes

16 from ruptured tendons are able to colonize these biode-

17 gradable scaffolds.

18 Methods Human primary tenocyte cultures of ruptured

19 quadriceps tendons were seeded on PU scaffolds. After 3,

20 10 and 15 days of incubation, the samples were stained

21 with haematoxylin and eosin and were examined under

22 white light microscopy. After 15 and 30 days of incuba-

23 tion, samples were examined under transmission electron

24 microscope. Total collagen accumulation was also evalu-

25 ated after 15, 30 and 45 days of culture.

26 Results After 15 and 30 days of culture, tenocyte-seeded

27 scaffolds showed cell colonization and cell accumulation

28around interconnecting micropores. Tenocyte phenotype

29was variable. Collagen accumulation in seeded scaffolds

30demonstrated a progressive increase after 15, 30 and

3145 days of culture, while control non-seeded scaffolds

32show no collagen accumulation.

33Conclusion These results showed that human tenocytes

34from ruptured quadriceps tendon can be seeded on

35polycaprolactone-based PU urea scaffolds and cultured for

36a long time period (45 days). This study also showed that

37human tenocytes from ruptured tendons seeded on PU

38scaffolds are able to penetrate the scaffold showing a

39progressively higher collagen accumulation after 15, 30

40and 45 days of incubation. This study provides the basis to

41use this PU biodegradable scaffold in vivo as an augmen-

42tation for chronic tendon ruptures and in vitro as a scaffold

43for tissue engineering construct.

44

45Keywords Tendon rupture � Human tenocytes �

46Tissue engineering � Scaffolds

47Introduction

48Primary disorders of tendons affect a cross-section of the

49population and are responsible for substantial morbidity

50both in sports and in the workplace [10].

51Tendon injuries range from acute traumatic tendon

52rupture to chronic overuse injuries [3]. Management of

53chronic tendon ruptures is challenging for the orthopaedic

54surgeon. The ends of chronic ruptured tendons are fre-

55quently retracted and have an atrophic appearance [20].

56When there is a big loss of tendon substance, the rup-

57tured tendon cannot be repaired and grafting is necessary.

58Tendon augmentation can be provided through autografts,

59allografts and synthetic grafts [12].

A1 L. Ruzzini (&) � U. G. Longo � S. Campi � V. Denaro

A2 Department of Orthopaedic and Trauma Surgery,

A3 Center for Integrated Research, Universita Campus Bio-Medico,

A4 Via A. Del Portillo, 200, Rome, Italy

A5 e-mail: l.ruzzini@unicampus.it

A6 L. Ruzzini

A7 Department of Orthopedics, Ospedale Pediatrico Bambino Gesu,

A8 Via Torre di Palidoro Palidoro, Rome, Italy

A9 N. Maffulli

A10 Centre for Sports and Exercise Medicine, Barts and The London

A11 School of Medicine and Dentistry Mile End Hospital,

A12 275 Bancroft Road, London E1 4DG, UK

A13 A. O. Muda

A14 Department of Pathology, Universita Campus Biomedico,

A15 Via A. Del Portillo, 200, Rome, Italy

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60 Many new tissue engineered materials have been

61 introduced: artificial polymers, biodegradable films and

62 biomaterials derived from animals or human, using a

63 combination of principles of engineering and biology [11].

64 The use of synthetic materials presents some advantages

65 on the autograft augmentation, such as the simplicity of the

66 technique and the absence of donor site morbidity [1].

67 Tendon engineering is the new frontier for tissue

68 replacement and regeneration. This approach utilizes

69 appropriate cell-scaffold construct to provide 3D templates

70 that support damaged tissue and allow growth of the

71 regenerate tissue. The rationale for using a scaffold device

72 for tendon repair may include mechanical augmentation,

73 improving the rate and quality of biological healing, or

74 both. Scaffolds with robust mechanical and suture retention

75 properties, applied in a surgically appropriate manner, may

76 have the ability to ‘‘off-load’’ the repair at time zero and for

77 some period of postoperative healing, depending on the

78 rate and extent of scaffold remodelling [13].

79 Polyurethanes (PU) are a large family of degradable

80 polymers of synthetic origin and are of great interest as

81 they offer a variety of possibilities due to their mechanical

82 properties in combination with a high degree of manufac-

83 tural reproducibility. Polyurethanes have a good biocom-

84 patibility and have been used in a variety of applications

85 such as ACL reconstruction, trapeziometacarpal joint

86 resurfacing and bone graft substitutes [5, 16, 21].

87 In this study, human tenocytes taken from ruptured

88 quadriceps tendons were seeded on the biodegradable PU

89 (Artelon, Artimplant AB, Hulda Mellgrens gata 5, SE-421

90 32 Vastra Frolunda, Sweden). Scaffold colonization and

91 collagen production after different culture periods were

92 analyzed to understand whether tenocytes from ruptured

93 tendons are able to colonize these biodegradable scaf-

94 folds. Artelon scaffold was previously demonstrated to

95 provide a high load to failure in Achilles tendon repair

96 cadaveric models [4] but the compatibility with tenocytes

97 was not investigated. This is the first in vitro study to

98 analyze tenocyte behaviour when seeded on PU (Artelon)

99 scaffold.

100 Materials and methods

101 Reagents

102 Dulbecco’s modified Eagle’s medium (DMEM) was pur-

103 chased from Lonza Group (Switzerland). All other reagents

104 were purchased from Sigma Chemical (St. Luis, USA)

105 unless stated otherwise.

106 Artelon scaffolds were gently provided by Artimplant

107 AB, Hulda Mellgrens gata 5, SE-421 32 Vastra Frolunda,

108 Sweden.

109Scaffold

110The scaffold used (Artelon, Artimplant) is a polycapro-

111lactone-based PU urea commercially available scaffold [5].

112This is a long-term degradable biomaterial, which degrades

113by hydrolysis over a period of approximately 5 years.

114Artelon (Artimplant) is a highly porous scaffold with

115interconnected pores. Each sample was cut into 10 9

11610 mm slides with a thickness of 2 mm.

117Cell culture

118The research protocol was approved by the Human

119Research Ethics committee of our institution.

120Primary human tenocytes were isolated from ruptured

121quadriceps tendons during surgical repair of the ruptured

122tendon.

123With written informed consent, three tendon tissue

124blocks (2 9 2 9 2 mm) were harvested from the quadri-

125ceps tendon of three patients affected by quadriceps tendon

126rupture. All patients were male amateur sportive (mean

127age 38).

128These tissue blocks were used as explants for primary

129cell cultures. The tendon samples were then washed with

130sterile PBS and cut into small pieces. The samples were

131then subjected to overnight collagenase (10 % w/v)

132digestion in DMEM containing 10 % (v/v) FBS. The

133digestion product was then centrifuged at 1,500 rpm for

13415 min, the supernatant was discharged, and the pellet was

135then re-suspended and cultured in 25-cm2 culture flasks in

136DMEM supplemented with 1 % pen-strep, 1 % Glut and

13710 % FBS at 37 �C in a humidified atmosphere of 5 % CO2

138in air. The medium was changed every 3 days until cells

139reached confluence. Cells were then harvested by trypsin-

140ization and centrifugation at 1,500 rpm for 5 min and were

141then re-suspended in DMEM with 1 % pen-strep, 1 % Glut

142and 10 % FBS and seeded to 75-cm2 culture flasks for

143subcultures.

144At confluence, cells were trypsinized and amplified for

145characterization and experimentation.

146Only cells from the second and third passages were used

147for the experiments to maintain a phenotype as close as

148possible to that occurring in vivo [23].

149Cells were cultured in 24-wells (105/well) on Artelon

150(Artimplant) in DMEM supplemented with 1 % pen-strep,

1511 % Glut and 10 % FBS and 50 lg/ml of ascorbic acid.

152For cell seeding, 1 9 105 cells mixed in 30 ll of

153medium were seeded over each scaffold and immedi-

154ately incubated for a 45-min period to allow cell

155attachment on the scaffold. Then, 1 ml of culture med-

156ium was added and the scaffolds were placed into the

157incubator (37 �C, 5 % CO2). Medium was changed

158every 3 days.

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159 Histology

160 After 3, 10 and 15 days of incubation, the tenocyte-seeded

161 Artelon scaffolds were placed in 20 ml of 10 % formalin in

162 a universal container for transportation to the Pathology

163 Department.

164 Then, the sample were dehydrated and embedded in

165 paraffin.

166 Once embedded in paraffin, the seeded scaffolds were

167 cut into 4 lm sections in a plane parallel to the long axis of

168 the scaffold.

169 Finally, sections were stained with haematoxylin and

170 eosin and were examined under white light microscopy.

171 For each sample, three slides were randomly selected

172 and examined using a light microscope. The identification

173 number on each slide was covered with a removable

174 sticker, and each slide was numbered using randomly

175 generated numbers. After one of the authors interpreted all

176 the slides once, the stickers were removed, a new sticker

177 was applied, and the slides were renumbered using a new

178 series of randomly generated numbers. Cellularity and cell

179 morphology were evaluated by the same author to verify

180 the drift of the tenocyte phenotype after seeding on the

181 Artelon scaffold and the two results were compared.

182 Transmission electron microscopy (TEM) evaluation

183 After 15 and 30 days of incubation, the tenocyte-seeded

184 Artelon scaffolds were fixed in 2.5 % glutaraldehyde, post-

185 fixed with 2 % osmium tetroxide, dehydrated with the

186 graded series of ethanol, passed through propyleneoxide,

187 and then embedded in araldite resin. Ultrathin sections

188 were double-stained with uranyl acetate and lead citrate

189 and examined under a transmission electron microscope

190 (Philips CM 10) and photographed.

191 Collagen accumulation

192 The quantification of total collagen tenocyte-seeded Art-

193 elon scaffolds was assessed after 15, 30 and 45 days of

194 incubation as previously described [19].

195 Tenocyte-seeded scaffolds were cultured adding 50

196 lg/ml of ascorbic acid in DMEM supplemented with 10 %

197 FBS, 1 % Glut and 1 % Pen-strep.

198 At the end of PEMF exposure, cells were fixed with

199 ethanol. Cell layers were then stained with 0.1 % Sirius red

200 F3BA in saturated picric acid for 18 h, after which excess

201 of Sirius red was removed by washing under running tap

202 water. The dye was then eluted with 0.1 N NaOH/methanol

203 (50:50), and the collagen quantitated by measuring spec-

204 trophotometrically at 490 nm. The values were then nor-

205 malized against total protein concentration. All assays

206were performed in triplicate. Non-seeded Artelon scaffolds

207were used as control.

208Statistical analysis

209Data are typical results from a minimum of three replicated

210experiments and are expressed as mean ± SD. Kappa

211statistics were used to assess the agreement between the

212cellularity and cell morphology of the slides. Variations in

213collagen expression over time in tenocyte-seeded scaffolds

214and non-seeded scaffolds were analyzed at each time point

215using paired Student’s t test. A p value less than 0.05 was

216considered significant.

217Results

218After 14 days of culture, tenocyte-seeded scaffolds showed

219cell colonization and cell accumulation around intercon-

220necting micropores. Collagen matrix production was also

221showed (eosinophylic matrix) (Fig. 1).

222Cell morphology was variable, showing both fibroblast-

223like appearance with spindle shape and flattened nuclei and

224flattened and polygonal shape with rounded nuclei (Fig. 2).

225Using the kappa statistics, the agreement between the two

226readings ranged from 0.61 to 0.85.

227Transmission electron microscopy (TEM) evaluation

228Ultrastructural examination showed tenocyte colonization

229of the scaffold after both 15 and 30 days of culture. A deep

230connection between the scaffold micropores and the cells

Fig. 1 The figure shows tenocyte accumulation around interconnect-

ing pores (circle) of the scaffold and collagen production after

15 days of culture. Magnification 940

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231 was demonstrated showing cytoplasmic processes going

232 through the micropores of the scaffold (Fig. 3).

233 Collagen accumulation

234 Collagen accumulation in seeded scaffolds demonstrated a

235 statistically significant progressively increase after 15, 30

236 and 45 days of culture (p\ 0.05), while control non-see-

237 ded scaffolds show no collagen accumulation (Table 1;

238 Fig. 4).

239 Seeded Artelon scaffolds showed a 29.5 % (p\ 0.05)

240 increase in collagen accumulation between 15 and 30 days

241 of culture and 29.2 % (p\ 0.05) increase between 30 and

242 45 days of culture, showing a doubling of collagen pro-

243 duction between 15 and 45 days of cell culture.

244 These results show that tenocytes increase collagen

245 production over time, demonstrating scaffold colonization

246 and capacity to survive on Artelon.

247We performed a post hoc power analysis on our results.

248With a total sample size of 9 specimens, our study has a

249power of 0.80 to detect a significant difference at 5 %

250significance level.

251Discussion

252The most important finding of the present study is that

253human tenocytes isolated from ruptured quadriceps tendons

254can be seeded on polycaprolactone-based PU urea scaffolds

255(Artelon) and cultured for a long time period.

256This study also showed that human tenocytes from

257ruptured tendons seeded on Artelon scaffolds are able to

258colonize the scaffold showing a progressively higher col-

259lagen accumulation after 15, 30 and 45 days of incubation.

260To our knowledge, this is the first study to investigate

261behaviour of human tenocytes seeded on Artelon scaffolds.

Fig. 2 The figure shows the variable shape of tenocytes cultured on the scaffold: spindle shaped with flattened nuclei (b) and polygonal shaped

with rounded nuclei (a, c, d). Magnification 940

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262 While there are concerns regarding the use of allografts

263 and autografts for immunogenicity and disease transmis-

264 sion and donor site pathology, the use of synthetic scaffolds

265 is increasing worldwide [2].

266 Since synthetic scaffolds were first used in the 1980s,

267 different materials were fabricated such as dacron, poly-

268 ester, carbon fibre, polypropylene, polyacrylamide, nylon

269 fabric and silicone [7, 9, 17, 18]. The advantage of syn-

270 thetic scaffolds is that they have superior mechanical

271 properties than biological scaffolds, even though their

272 biocompatibility is lower [15].

273 Tendon grafts are required to be made of flexible

274 and elastic materials that allow adaptation of the autoge-

275 nous tissue formed. The use of degradable elastomers is

276widespread, because of their capacity to act as a temporary

277reinforcement during tendon healing; moreover, it is possi-

278ble to control their mechanical properties, degradation rates

279and structure [5]. Among synthetic biodegradable materials,

280polyurethane (PU) polymers are of great interest due to their

281biocompatibility with soft tissue. They are degradable syn-

282thetic polymers well known for their mechanical properties

283in combination with a high degree of manufactural repro-

284ducibility [6].

285Artelon scaffold was recently demonstrated to have

286good mechanical properties when used as an augmentation

287for Achilles tendon repair showing strong resistance to

288failure [4].

289Henry et al. [6] showed that the polyesterurethane

290scaffolds have a significantly smaller chronic inflammatory

291reaction than bovine pericardium graft in a subcutaneous

292rabbit implant model.

293In vitro studies showed that Artelon scaffolds are opti-

294mal substrates for cell seeding [5, 21, 22]. In fact mono-

295nuclear cells showed a lower apoptotic and necrotic rate

296when seeded on polyurethane polymers than other sub-

297strates [5]. Moreover, Siepe et al. [21, 22] demonstrate that

298also myocardial myoblast can be seeded on Artelon scaf-

299folds, showing an homogenous distribution throughout the

300scaffold. Artelon cell seeded implants were also showed to

301be good tissue regenerating scaffolds in in vivo studies

302demonstrating optimal biocompatibility in rat myocardial

303regeneration and in human dermis regeneration [8, 22].

Fig. 3 TEM evaluation shows a deep interaction between scaffold

and tenocytes showing cytoplasmic processes going into the scaffold

micropores. Magnification 95,000

Table 1 The chart shows collagen accumulation expressed as absorption at 490 nM normalized against total protein concentration in seeded and

non-seeded (control) scaffolds

14 days 30 days 45 days

Seeded scaffold 0.02425 ± 0.0028 0.03375 ± 0.0049 0.048 ± 0.0043

Non-seeded scaffold (control) 0.0065 ± 0.0021 0.0065 ± 0.0018 0.0065 ± 0.0025

Values are expressed as average ± SD

Fig. 4 The chart shows the increasing of collagen production of

tenocytes seeded on the Artelon scaffolds after 15, 30 and 45 days of

culture

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304 In our study, a polycaprolactone-based PU urea scaf-

305 folds (Artelon) with a porous architecture that allows cells

306 to penetrate the structure increasing the surface area over

307 which they can proliferate was used. Human tenocytes can

308 be seeded on Artelon implants showing scaffold coloni-

309 zation and collagen production in a long-term culture

310 period.

311 There is a rational basis for further investigations on the

312 use of Artelon implant as an engineered tendon replace-

313 ment. Tenocytes from ruptured quadriceps tendon were

314 used, demonstrating that they are able to colonize the

315 scaffold and to survive and produce collagen.

316 In this way, Artelon acts both as a bridge between the

317 free ends of the ruptured tendon and as a scaffold in which

318 tenocytes can penetrate and produce collagen.

319 We are fully aware of the limitations of this study.

320 Shortcomings of this study include the fact that we did not

321 assess cell viability, gene expression and we did not per-

322 form collagen typing experiments. Moreover, there are

323 limitations to extrapolating in vitro findings to clinical

324 situations, as many other factors interact in the human

325 body, including inflammatory reactions, circulation, and

326 other cytokines and growth factors.

327 The lack of a cell proliferation assay did not let to

328 understand whether the increase of collagen production is

329 real or is due to the increase of cell proliferation. Another

330 limitation of our study is that we did not perform a collagen

331 typing evaluation; even though we would expect greater

332 quantities of type 3 collagen form tenocyte from ruptured

333 tendons [14], it would be interesting to evaluate whether

334 tenocytes from ruptured tendons seeded on Artelon scaf-

335 folds are able to drift collagen production from type 3

336 (typically produced by tenocytes from ruptured tendons) to

337 type 1 (typically produced by tenocytes from intact

338 tendons).

339 We are also aware that the tendon samples were taken

340 only from ruptured quadriceps tendon. In this respect, it

341 would be interesting to evaluate tenocytes from different

342 tendons and to compare tenocytes from ruptured and

343 healthy tendons. We acknowledge that these are only

344 preliminary analyses on the tenocyte biocompatibility with

345 Artelon scaffold, assessing only collagen production and

346 microscopic/ultrastructural evaluation. These data can

347 therefore be taken only as an early evidence for a possible

348 use of polycaprolactone-based PU urea scaffolds (Artelon)

349 as an augmentation or a substrate for a tissue engineering

350 approach for the management of large tendon defects.

351 Conclusion

352 The basis for the enhancement of application of synthetic

353 biodegradable polyurethane (PU) polymers in vivo as

354tendon grafts for the augmentation of tendon repair in

355chronic tendon ruptures and in vitro as cell-seeded scaffolds

356to improve the healing of ruptured tendons as a engineered

357tendon construct are provided by the present study.

358However, further clinical and in vitro investigations are

359necessary to better evaluate the validity of Artelon scaffold

360both as a tendon graft and as a 3D tenocyte seeded con-

361struct for tendon engineering.

362Conflict of interest The authors declare no conflict of interest.

363References

3641. Altman GH, Horan RL, Lu HH, Moreau J, Martin I, Richmond365JC, Kaplan DL (2002) Silk matrix for tissue engineered anterior366cruciate ligaments. Biomaterials 23:4131–41413672. Chen J, Xu J, Wang A, Zheng M (2009) Scaffolds for tendon and368ligament repair: review of the efficacy of commercial products.369Expert rev med dev 6:61–733703. Denaro V, Ruzzini L, Longo UG, Franceschi F, De Paola B,371Cittadini A, Maffulli N, Sgambato A (2010) Effect of dihydro-372testosterone on cultured human tenocytes from intact supraspi-373natus tendon. Knee Surg Sports Traumatol Arthrosc 18:971–9763744. Giza E, Frizzell L, Farac R, Williams J, Kim S (2011) Aug-375mented tendon Achilles repair using a tissue reinforcement376scaffold: a biomechanical study. Foot Ankle Int 32:S545–S5493775. Gretzer C, Gisselfalt K, Liljensten E, Ryden L, Thomsen P (2003)378Adhesion, apoptosis and cytokine release of human mononuclear379cells cultured on degradable poly(urethane urea), polystyrene and380titanium in vitro. Biomaterials 24:2843–28523816. Henry JA, Burugapalli K, Neuenschwander P, Pandit A (2009)382Structural variants of biodegradable polyesterurethane in vivo383evoke a cellular and angiogenic response that is dictated by384architecture. Acta biomat 5:29–423857. Hunter JM, Singer DI, Jaeger SH, Mackin EJ (1988) Active386tendon implants in flexor tendon reconstruction. J Hand Surg38713:849–8593888. Huss FR, Nyman E, Gustafson CJ, Gisselfalt K, Liljensten E,389Kratz G (2008) Characterization of a new degradable polymer390scaffold for regeneration of the dermis: in vitro and in vivo391human studies. Organogenesis 4:195–2003929. Kain CC, Manske PR, Reinsel TE, Rouse AM, Peterson WW393(1988) Reconstruction of the digital pulley in the monkey using394biologic and nonbiologic materials. J Orthop Res 6:871–87739510. Longo UG, Franceschi F, Ruzzini L, Rabitti C, Morini S, Maffulli396N, Forriol F, Denaro V (2007) Light microscopic histology of397supraspinatus tendon ruptures. Knee Surg Sports Traumatol398Arthrosc 15:1390–139439911. Longo UG, Lamberti A, Maffulli N, Denaro V (2010) Tendon400augmentation grafts: a systematic review. Br Med Bull 94:165–40118840212. Longo UG, Lamberti A, Maffulli N, Denaro V (2010) Tissue403engineered biological augmentation for tendon healing: a sys-404tematic review. Br Med Bull 98:31–5940513. Longo UG, Lamberti A, Rizzello G, Maffulli N, Denaro V (2012)406Synthetic augmentation in massive rotator cuff tears. Med Sports407Sci 57:168–17740814. Maffulli N, Ewen SW, Waterston SW, Reaper J, Barrass V409(2000) Tenocytes from ruptured and tendinopathic achilles ten-410dons produce greater quantities of type III collagen than teno-411cytes from normal achilles tendons. An in vitro model of human412tendon healing. Am J Sports Med 28:499–505

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413 15. Maffulli N, Longo UG, Denaro V (2010) Novel approaches for the414 management of tendinopathy. J Bone Joint SurgAm92:2604–2613415 16. Nilsson A, Liljensten E, Bergstrom C, Sollerman C (2005)416 Results from a degradable TMC joint Spacer (Artelon) compared417 with tendon arthroplasty. J Hand Surg 30:380–389418 17. Ozaki J, Fujimoto S, Masuhara K, Tamai S, Yoshimoto S (1986)419 Reconstruction of chronic massive rotator cuff tears with syn-420 thetic materials. Clin Orth Rel Res 202:173–183421 18. Post M (1985) Rotator cuff repair with carbon filament. A pre-422 liminary report of five cases. Clin Orth Rel Res 196:154–158423 19. Scutt N, Rolf CG, Scutt A (2006) Glucocorticoids inhibit teno-424 cyte proliferation and Tendon progenitor cell recruitment. J Ort-425 hop Res 24:173–182426 20. Sharma P, Maffulli N (2008) Tendinopathy and tendon injury: the427 future. Disabil Rehabil 30:1733–1745

42821. Siepe M, Giraud MN, Liljensten E, Nydegger U, Menasche P,429Carrel T, Tevaearai HT (2007) Construction of skeletal myoblast-430based polyurethane scaffolds for myocardial repair. Art org 31:431425–43343222. Siepe M, Giraud MN, Pavlovic M, Receputo C, Beyersdorf F,433Menasche P, Carrel T, Tevaearai HT (2006) Myoblast-seeded434biodegradable scaffolds to prevent post-myocardial infarction435evolution toward heart failure. J Thorac Cardiovasc Surg 132:436124–13143723. Yao L, Bestwick CS, Bestwick LA, Maffulli N, Aspden RM438(2006) Phenotypic drift in human tenocyte culture. Tissue Eng43912:1843–1849

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