Decellularized Human Dermis to Treat Massive Rotator Cuff Tears: In Vitro Evaluations

Interest is increasing in biological scaffolds for tissue regeneration such as extracellular matrix membranes, developed through soft tissue decellularization. Extracellular matrix membranes were developed to heal different tendon and soft tissue lesions that are very frequent in the general population with high health-care costs and patient morbidity. The aim of this research was to evaluate a human dermal matrix (HDM) decellularized by a chemico-physical method. A primary culture of rat tenocytes was performed: tenocytes were seeded on HDM samples and on polystyrene wells as controls (CTR). Cell viability and synthetic activity were evaluated at 3 and 7 days. An in vitro microwound model was used to evaluate HDM bioactivity: after tenocyte expansion, artificial wounds were created, HDM extracts were added, and closure time and decorin synthesis were monitored histomorphometrically at 1, 4, 24, and 72 hr. A significant higher amount of collagen I was observed when cells were cultured on HDM in comparison with that on CTR (3 days: p < 0.0001; 7 days: p < 0.05). In HDM group, fibronectin synthesis was significantly higher at both experimental times (p < 0.0001). At 3 days, proteoglycans and transforming growth factor-β1 releases were significantly higher on HDM (p < 0.0001 and p < 0.005, respectively). The artificial microwound closure time and decorin expression were significantly enhanced by the addition of 50% HDM extract (p < 0.05). In vitro data showed that the decellularization technique enabled the development of a matrix with adequate biological and biomechanical properties.     

In vivo tendon engineering with skeletal muscle derived cells in a mouse model

Engineering a functional tendon with strong mechanical property remains an aim to be achieved for its eventual application. Both skeletal muscle and tendon are closely associated during their development and both can bear strong mechanical loading dynamically. This study explored the possibility of engineering stronger tendons with mouse skeletal muscle derived cells (MDCs) and with mouse tenocytes as a control. The results demonstrated that both MDCs and tenocytes shared the gene expression of growth differentiation factor-8 (GDF-8), collagens I, III, VI, scleraxis and tenomodulin, but with MyoD gene expression only in MDCs. Quantitatively, MDCs expressed higher levels of GDF-8, collagens III and VI (p < 0.05), whereas tenocytes expressed higher levels of collagen I, scleraxis and tenomodulin (p < 0.05). Interestingly, MDCs proliferated faster with more cells in S + G2/M phases than tenocytes (p < 0.05). After been seeded on polyglycolic acid (PGA) fibers, MDCs formed better quality engineered tendons with more mature collagen structure and thicker collagen fibrils as opposed to tenocyte engineered tendons. Biochemically, more collagen VI and decorin were produced in the former than in the later. Functionally, MDC engineered tendons exhibited stronger mechanical properties than tenocyte engineered tendons, including maximal load, stiffness, tensile strength and Young's modulus (p < 0.05). Furthermore, with the increase of implantation time, MDCs gradually lost their expression of myogenic molecules of MyoD and desmin and gained the expression of tenomodulin, a marker for tenocytes. Collectively, these results indicate that MDCs may serve as a desirable alternative cell source for engineering functional tendon tissue.     

Phenotypic drift in human tenocyte culture

Tendon ruptures are increasingly common, repair can be difficult, and healing is poorly understood. Tissue engineering approaches often require expansion of cell numbers to populate a construct, and maintenance of cell phenotype is essential for tissue regeneration. Here, we characterize the phenotype of human Achilles tenocytes and assess how this is affected by passaging. Tenocytes, isolated from tendon samples from 6 patients receiving surgery for rupture of the Achilles tendon, were passaged 8 times. Proliferation rates and cell morphology were recorded at passages 1, 4, and 8. Total collagen, the ratio of collagen types I and III, and decorin were used as indicators of matrix formation, and expression of the integrin beta1 subunit as a marker of cell-matrix interactions. With increasing passage number, cells became more rounded, were more widely spaced at confluence, and confluent cell density declined from 18,700/cm2 to 16,100/cm2 ( p = 0.009). No change to total cell layer collagen was observed but the ratio of type III to type I collagen increased from 0.60 at passage 1 to 0.89 at passage 8 ( p < 0.001). Decorin expression significantly decreased with passage number, from 22.9 +/- 3.1 ng/ng of DNA at passage 1, to 9.1 +/- 1.8 ng/ng of DNA at passage 8 ( p < 0.001). Integrin expression did not change. We conclude that the phenotype of tenocytes in culture rapidly drifts with progressive passage.     

Human hamstring tenocytes survive when seeded into a decellularized porcine Achilles tendon extracellular matrix

Abstract

Tendon ruptures and defects remain major orthopaedic challenges. Tendon healing is a time-consuming process, which results in scar tissue with an altered biomechanical competence. Using a xenogeneic tendon extracellular matrix (ECM) as a natural scaffold, which can be reseeded with autologous human tenocytes, might be a promising approach to reconstruct damaged tendons. For this purpose, the porcine Achilles (AS) tendons serving as a scaffold were histologically characterized in comparison to human cell donor tendons. AS tendons were decellularized and then reseeded with primary human hamstring tenocytes using cell centrifuging, rotating culture and cell injection techniques. Vitality testing, histology and glycosaminoglycan/DNA quantifications were performed to document the success of tendon reseeding. Porcine AS tendons were characterized by a higher cell and sulfated glycosaminoglycan content than human cell donor tendons. Complete decellularization could be achieved, but led to a wash out of sulfated glycosaminoglycans. Nevertheless, porcine tendon could be recellularized with vital human tenocytes. The recellularization led to a slight increase in cell number compared to the native tendon and some glycosaminoglycan recovery. This study indicates that porcine tendon can be de- and recellularized using adult human tenocytes. Future work should optimize cell distribution within the recellularized tendon ECM and consider tendon- and donor species-dependent differences.     

Development and characterization of an acellular human pericardial matrix for tissue engineering

This study aimed to produce an acellular human tissue scaffold with a view to recellularization with autologous cells to produce a tissue-engineered pericardium that can be used as a patch for cardiovascular repair. Human pericardia from cadaveric donors were treated sequentially with hypotonic buffer, SDS in hypotonic buffer, and a nuclease solution. Histological analysis of decellularized matrices showed that the human pericardial tissue retained its histioarchitecture and major structural proteins. There were no whole cells or cell fragments. There were no significant differences in the hydroxyproline (normal and denatured collagen) and glycosaminoglycan content of the tissue before and after decellularization (p > 0.05). There were no significant changes in the ultimate tensile strength after decellularization (p > 0.05). However, there was an increased extensibility when the tissue strips were cut parallel to the visualized collagen bundles (p = 0.005). No indication of contact or extract cytotoxicity was found when using human dermal fibroblasts and A549 cells. In summary, successful decellularization of the human pericardium was achieved producing a biocompatible matrix that retained the major structural components and strength of the native tissue.     

Method of preparing a decellularized porcine tendon using tributyl phosphate

Extracellular matrix (ECM) materials are currently utilized for soft tissue repair applications such as vascular grafts, tendon reconstruction, and hernia repair. These materials are derived from tissues such as human dermis and porcine small intestine submucosa, which must be rendered acellular to prevent disease transmission and decrease the risk of an immune response. The ideal decellularization technique removes cells and cellular remnants, but leaves the original collagen architecture intact. The tissue utilized in this study was the central tendon of the porcine diaphragm, which had not been previously investigated for soft tissue repair. Several treatments were investigated during this study including peracetic acid, TritonX-100, sodium dodecyl sulfate, and tri(n-butyl) phosphate (TnBP). Of the decellularization treatments investigated, only 1% TnBP was effective in removing cell nuclei while leaving the structure and composition of the tissue intact. Overall, the resulting acellular tissue scaffold retained the ECM composition, strength, resistance to enzymatic degradation, and biocompatibility of the original tissue, making 1% TnBP an acceptable decellularization treatment for porcine diaphragm tendon.     

HLA DPB1*0201 allele is negatively associated with immunoglobulin E responsiveness specific for house dust mite allergens in Taiwan

BACKGROUND: House dust mite (HDM) Dermatophagoides pteronyssinus is the most important source of indoor allergens that cause allergic diseases in Taiwan. We prepared purified HDM allergens (Der p 1, Der p 2 and Der p 5) to detect allergen-specific immunoglobulin (Ig) E responsiveness among a large number of test subjects. The robust genetic typing system for HLA class II genes also facilitated the study on association of HLA and allergic response toward HDM. OBJECTIVE: This study intended to investigate the association between HLA class II alleles and the IgE responsiveness to the major allergens from HDM, D. pteronyssinus. METHODS: Two hundred and forty-eight subjects were selected for HLA association study. Plasma HDM allergen (Der p 1, Der p 2, Der p 5) -specific IgE and Der p 2-specific IgG antibodies were detected by ELISA, while HLA class II -DRB1, -DQA1, -DQB1, -DPB1 genetic polymorphism was determined by polymerase chain reaction/sequence-specific oligonucleotide probe hybridization (PCR/SSOPH). Statistical comparison of the allelic distribution of each HLA class II genes among the individuals with/without HDM allergen-specific IgE and IgG antibodies were performed. RESULTS: There was no significant association between HLA DRB1, DQB1, DQA1 alleles and HDM-specific IgE responsiveness noted. Only DRB1*0803 and the linked DQA1*0103 alleles showed positive association with Der p 5-specific IgE responsiveness. However, we found that HLA-DPB1*1301 predisposed subjects to IgE responsiveness to HDM Der p 5. HLA DPB1*0501 was weakly associated with the IgE responsiveness to HDM Der p 1 and Der p 5. There was a strong negative association between the HLA-DPB1*0201 allele with IgE responsiveness to Der p 1 (OR: 0.30, P 相似文献   

Biochemical changes caused by decellularization may compromise mechanical integrity of tracheal scaffolds

Tissue-engineered airways have achieved clinical success, but concerns remain about short-term loss of biomechanical properties, necessitating a stent. This study investigated the effect of chemical–enzymatic decellularization on biochemical properties of trachea important for cell attachment and vascularization (fibronectin and laminin) and cartilage matrix homeostasis (type II collagen and glycosaminoglycans (GAG)), as well as biomechanical status. Native trachea was used as a control, and NDC trachea stored in phosphate buffered saline (PBS) in parallel to decellularization was used as a time-matched control. Decellularization removed most cells, but chondrocytes and DNA remained after 25 cycles. Fibronectin was retained throughout the lamina propria and laminin at basement membranes. DNA accumulation along ECM fibres was seen. A decline in soluble collagen was observed in decellularized tissue. GAG content of cartilage rings was reduced, even in PBS control tissue from 20 cycles onwards (p < 0.05), but decellularization caused the greatest loss (p < 0.01). Tensile strength declined throughout the process, but was significant only at later time points. The data demonstrate that the substantial reduction in GAG might contribute to loss of mechanical integrity of biotracheas. Overcoming structural changes that cause an imbalance in cartilage matrix equilibrium will be necessary to optimize clinical benefit, enabling widespread use of biotracheas.     

Effect of Decorin and Dermatan Sulfate on the Mechanical Properties of a Neocartilage

Decorin is known to influence the size of collagen fibrils in ligaments and tendons and it has been hypothesized to provide a structural link between collagen fibrils in connective tissues, including cartilage. Coincidently, mechanical properties of skin, ligament, and tendons are altered in decorin knockout mice, suggesting it may influence the structural properties of tissue or tissue matrix organization. To further examine the role of decorin in the extracellular matrix development and subsequent material properties of cartilage, tissue (neocartilage) was grown in a 3D culture model using a pure population of genetically modified chondrocytes stably overexpressing decorin (DCN) or decorin lacking dermatan sulfate (MDCN). An empty vector (CON) served as a virus control. Following generation of the cartilage-like tissues, mechanical properties in tension and compression, collagen fibril diameter, matrix organization, and biochemistry of the tissue were determined. There were no differences between CON and DCN tissues in any parameter measured. In contrast, tissue generated in MDCN cultures was thinner, had higher collagen density, and higher elastic moduli as compared to both CON and DCN tissues. Considering there was no difference in stiffness between CON and DCN tissues, the notion that decorin contributes to the mechanical properties via load transfer was refuted in this model. However, contrasts in the mechanical properties of the MDCN tissue suggest that the dermatan sulfate chains on decorin influences the organization/maturation and resultant mechanical properties of the matrix by as an yet-unidentified regulatory mechanism.     

组织块法培养成年兔肌腱细胞

背景：体外分离培养肌腱细胞,熟悉其生物学特性是研究肌腱愈合机制、改善肌腱愈合内环境的前提和基础。 目的：采用组织块法体外培养成年兔肌腱细胞,观察其细胞形态、生长增殖情况以及Ⅰ型、Ⅲ型胶原蛋白的表达。 方法：无菌条件下取成年新西兰白兔双侧后肢趾屈肌腱,手术显微镜下剥离、去除肌腱外膜组织,剪成组织小块,0.25%胰蛋白酶和0.1%胶原酶Ⅰ消化10~15 min,离心去上清,将组织小块转移到培养瓶中,贴壁后加入1 mL培养液,待细胞游出贴壁生长时,再加培养液继续培养,每3 d换液1次,当细胞长到80%~90%融合时按1∶3传代。 结果与结论：一般10 d左右细胞会从组织块游出贴壁生长,此时细胞多呈星形或不规则形,随着时间延长细胞逐渐增多,呈梭形的成纤维细胞样。传代细胞刚接种时圆形,4~6 h开始贴壁并伸展为梭形,排列逐渐规则成群。从传代细胞的生长曲线可看出,前4 d细胞生长较慢,为潜伏期,第5天后进入快速增殖期,7 d以后进入平台期。免疫荧光法证实Ⅰ型胶原染色阳性,而Ⅲ型胶原染色呈阴性。结果表明采用组织块法可在体外成功培养成年兔肌腱细胞。     

Human mesenchymal progenitor cell responses to a novel textured poly(L-lactide) scaffold for ligament tissue engineering

Biocompatibility and cell seeding capability of a new cell scaffold made of textured polylactic acid (PLA) fibers was investigated as a new material for tissue engineering of anterior cruciate ligaments (ACL). Adhesion and proliferation of human mesenchymal progenitor cells (MPC) was investigated after 15 days by scanning electron microscopy and standard histology. Expression of collagen type I and III, fibronectin, tenascin C, decorin, smooth muscle actin, and the matrix metalloproteinases MMP-1 and MMP-2, as well as their tissue inhibitors TIMP-1 and TIMP-2 was analyzed using real-time PCR. Protein expression of collagen I and III, tenascin C, and proliferating nuclear antigen (PCNA) was determined by immunohistology. Apoptosis was analyzed by detection of p53 expression and TUNEL staining. MPC seeded the scaffold homogeneously and showed good cell growth and no increased rate of apoptosis. After 15 days, the matrix forming genes collagen type I, tenascin C, and decorin were upregulated, indicating the formation of a ligament-like matrix. MMP-1 and TIMP-1 were also significantly increased, suggesting initial matrix remodeling. It was concluded that the new porous PLA scaffold allowed homogeneous cell seeding, a fibroblastic phenotype and the production of a ligament-like matrix and, therefore, might be a suitable cell carrier for ACL tissue engineering.     

Association between mite allergen (Der p 1, Der f 1, Blo t 5) levels and microscopic identification of mites or skin prick test results in asthmatic subjects

BACKGROUND: Mite allergens have been involved in airway sensitization and allergic diseases. Immunoassays for the identification and quantifiction of house dust mite (HDM) allergens are useful to improve the knowledge of regional mite fauna and the remediation of mite allergens in allergic diseases. The present study analyzed the association between levels of HDM allergen and results of mite identification or skin prick test (SPT) in two different areas of Bahia, Brazil. METHODS: Forty-two asthmatic subjects from a rural area (group I; n = 21) and a slum (group II; n = 21) were evaluated through SPT with HDM allergens and had dust samples collected at their homes for mite identification and allergen measurements. RESULTS: Positive SPT to Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis allergens were observed in 42.9, 38.0 and 42.9% subjects from group I and in 47.6, 19.0 and 33.3% subjects from group II, respectively. D. pteronyssinus and B. tropicalis were identified in approximately 76 and 50% of samples from both groups, respectively. D. farinae was identified in 38.0 and 9.5% of samples from groups I and II, respectively (p < 0.005). Der p 1, Der f 1 and Blo t 5 detection were associated with mite identification (p < 0.05). Association between HDM allergen levels over 2 microg/g of dust and positive SPT occurred only with D. pteronyssinus (p < 0.0001). CONCLUSIONS: D. pteronyssinus was the most prevalent mite species in this study followed by B. tropicalis and D. farinae. Immunoassays done to measure mite allergens were associated with mite-species identification. We conclude that these three mite species must be included on panels for the diagnosis of allergic airway diseases in subjects living in such regions.     

Lung development following diaphragmatic hernia in the fetal rabbit

Diaphragmatic hernia was created in 39 rabbit fetuses on day 23 of gestation. Fifteen fetuses underwent a sham thoracotomy (SHAM). Thirty-nine non-operated littermates served as internal controls (CTR). Fetuses were harvested by Caesarean section on days 25, 27, 29 and 30 of gestation. Pulmonary response was evaluated by lung to body weight ratio (LBWR), morphometry, and density of type II pneumocytes. No difference was found between CTR and SHAM fetuses at term. CDH fetuses had smaller lungs (LBWR 0.014 +/- 0.004 versus 0.030 +/- 0.04 in CTR, P < 0.0001), a less complex acinus [mean terminal bronchial density (MTBD) 1.786 +/- 0.408 versus 0.917 +/- 0. 188, P < 0.0001], thicker alveolar septa [mean wall transection length (LMW) 0.0221 +/- 0.008 versus 0.0142 +/- 0.002, P = 0.0003], and a lower type II cell count (144.5 +/- 19.33 versus 216.2 +/- 27.85 per high power field, P < 0.0001). The differences in MTBD and LMW were significant from gestational day 25 onwards, and the differences in type II cell count from day 27 onwards. Surgical diaphragmatic hernia in rabbit fetuses in the late pseudoglandular phase reproduces many features of the pulmonary hypoplasia associated with human congenital diaphragmatic hernia, including the delayed maturation. The effects are present within 2 days following experimental diaphragmatic hernia and progress over time.     

The cross-talk between transforming growth factor-beta1 and ultrasound stimulation during mechanotransduction of rat tenocytes

Ultrasound is an effective noninvasive treatment for various tendinopathies. However, how tenocytes convert ultrasound stimulation into cascades of cellular and molecular events is not well understood. The purpose of this study is to elucidate the signaling pathways of tenocytes during ultrasound stimulation.

Primary cultures of tenocytes were harvested from Achilles tendons of Sprague–Dawley rats. The viability and proliferation of tenocytes, their genes expression, and the signaling pathways after ultrasound treatment with or without specific inhibitors were evaluated and analyzed.

The results showed that ultrasound treatment (100 mW/cm² for 20 min) significantly enhanced matrix metalloproteinase 13 (MMP-13), c-Fos, and c-Jun gene expression, increased JNK and p38, but not extracellular signal-regulated kinase-1/2 (ERK1/2), phosphorylation at 5 min, and sustained up to 60 min. JNK inhibitor and p38 inhibitor, but not ERK1/2 inhibitor, attenuated ultrasound-dependent induction of MMP-13 expression, indicating that the JNK and p38 pathways are required for ultrasound-induced MMP-13 expression in tenocytes. We also found that SB431542 (transforming growth factor-beta (TGF-β) receptor kinases inhibitor) suppressed ultrasound-induced MMP‐13 and c-Fos gene expression, and p38 phosphorylation.

This study revealed that ultrasound treatment stimulates tenocytes proliferation and regulates their matrix metabolism through the cross-talk between TGF-β and ultrasound-induced mitogen-activated protein kinases (MAPKs) signaling pathways.     

Preservation of aortic root architecture and properties using a detergent-enzymatic perfusion protocol

Aortic valve degeneration and dysfunction is one of the leading causes for morbidity and mortality. The conventional heart-valve prostheses have significant limitations with either life-long anticoagulation therapeutic associated bleeding complications (mechanical valves) or limited durability (biological valves). Tissue engineered valve replacement recently showed encouraging results, but the unpredictable outcome of tissue degeneration is likely associated to the extensive tissue processing methods. We believe that optimized decellularization procedures may provide aortic valve/root grafts improved durability. We present an improved/innovative decellularization approach using a detergent-enzymatic perfusion method, which is both quicker and has less exposure of matrix degenerating detergents, compared to previous protocols. The obtained graft was characterized for its architecture, extracellular matrix proteins, mechanical and immunological properties. We further analyzed the engineered aortic root for biocompatibility by cell adhesion and viability in vitro and heterotopic implantation in vivo. The developed decellularization protocol was substantially reduced in processing time whilst maintaining tissue integrity. Furthermore, the decellularized aortic root remained bioactive without eliciting any adverse immunological reaction. Cell adhesion and viability demonstrated the scaffold's biocompatibility. Our optimized decellularization protocol may be useful to develop the next generation of clinical valve prosthesis with a focus on improved mechanical properties and durability.     

脱细胞技术在组织工程血管中的应用进展

将天然血管经过脱细胞处理得到的脱细胞血管,被认为是一种具有广阔应用前景的组织工程血管支架材料.截至目前,细胞外基质(ECM)支架的制备方法仍缺乏统一标准.脱细胞方法的选择取决于组织来源和基质支架的用途,尤其对于脱细胞血管等需要长期承受血流冲击的基质支架材料来说,脱细胞方案的选择至关重要.细胞清除效率和细胞外基质支架的性...     

Decorin 基因转染抑制高糖培养的大鼠肾系膜细胞细胞外基质mRNA表达

目的： 观察核心蛋白聚糖Ⅱ（DCN）基因转染对高糖培养的大鼠肾系膜细胞（RMCs）细胞外基质（ECM）mRNA表达的影响。方法： 用已构建的AD-DCN腺病毒感染高糖环境培养的大鼠肾系膜细胞，采用半定量RT-PCR测定decorin、转化生长因子β₁（TGF-β₁）、纤维粘连蛋白（fibronectin）和IV型胶原（collagen IV） mRNA水平。结果： 高糖培养的RMCs感染AD-DCN后，DCN mRNA水平显著增高，24 h至第7 d，该组DCN mRNA水平始终显著高于高糖组及AD－LacZ感染组(P<0.05)，decorin mRNA水平增高后表现出对TGF-β₁ mRNA 水平的抑制作用，在mRNA水平，AD-DCN感染的RMCs细胞外基质fibronectin和collagen IV随TGF-β₁ mRNA水平下降而下降。结论： DCN重组腺病毒感染大鼠肾系膜细胞后能高效表达目的基因decorin并拮抗 TGF-β₁ 的生物活性，其下调ECM成分 mRNA水平的作用与使用TGF-β抗体的效果相似。     

Effects of house dust mite avoidance measures on Der p 1 concentrations and clinical condition of mild adult house dust mite-allergic asthmatic patients, using no inhaled steroids.

BACKGROUND: Exposure to house dust mite (HDM) allergens often results in worsening of asthma. Therefore, avoidance of exposure to HDM allergens is often proposed. Unfortunately, the most effective and feasible avoidance strategy is still not completely assessed. Consequently, we investigated the effects of a combined HDM avoidance strategy on HDM allergen concentrations and clinical condition of allergic, mild asthmatic, patients using no inhaled steroids. METHODS: Asthmatic patients, allergic to HDM, using no inhaled corticosteroids, were randomly allocated to an active (n = 76) or a placebo allergen-avoidance group (n = 81). Avoidance measures consisted of applying Acarosan(R) (placebo: water) to the living room and bedroom floors, and the use of HDM-impermeable covers for mattresses and bedding (placebo: cotton covers for mattresses only). Effects on allergen concentrations (Der p 1), FEV1, bronchial hyperresponsiveness, peak flow parameters and asthma symptom scores were studied during 20 weeks and controlled for the allergic status of the patients. RESULTS: The active covers reduced Der p 1 concentrations to 9.4% (P = 0.0001), and were always significant lower than in the placebo group (P = 0.0002). Acarosan(R) resulted in slight but significant decreases (twofold, P = 0.0001), both on living room and bedroom floors, but concentrations were never significantly lower than the placebo group. Although the combined avoidance strategy resulted in a considerable reduction in allergen load in the active group, no differences were seen between the two groups in any of the clinical parameters during the follow-up period in this group of allergic asthmatics, using no inhaled corticosteroids. Corrections for the allergic status did not alter these results. CONCLUSIONS: The combined avoidance strategy was effective in reducing HDM allergen concentration. This was especially achieved by the allergen-impermeable covers, while the effects of Acarosan(R) were only marginal. However, this allergen reduction was not reflected in a convincing improvement in clinical condition in this group of mild allergic asthmatics, using no inhaled steroids. Perhaps, a longer follow-up period would have resulted in more pronounced effects.     

Repair of tendon defect with dermal fibroblast engineered tendon in a porcine model

Harvesting autologous tenocytes for tendon engineering may cause secondary tendon defect at the donor site. Dermal fibroblasts are an easily accessible cell source and do not cause major donor site defect. This study aims to explore the possibility of tendon engineering using dermal fibroblasts. A total of 45 hybrid pigs were randomly divided into three groups: experimental group (n = 15)--repair of tendon defect with a dermal fibroblast engineered tendon; control group 1 (n = 15)--repair of defect with a tenocyte engineered tendon; and control group 2 (n = 15)-repair of defect with a scaffold alone. Both autologous dermal fibroblasts and tenocytes were seeded on polyglycolic acid (PGA) unwoven fibers to form a cell-scaffold construct and cultured in vitro for 7 days before in vivo implantation to repair a defect of flexor digital superficial tendon. Specimens were harvested at weeks 6, 14, and 26 for gross, histological, and mechanical analyses. Microscopy revealed good attachment of both dermal fibroblasts and tenocytes on PGA fibers and matrix production. In vivo results showed that fibroblast and tenocyte engineered tendons were similar to each other in their gross view, histology, and tensile strength. At 6 weeks, parallel collagen alignment was observed at both ends, but not in the middle in histology, with more cellular components than natural tendons. At weeks 14 and 26, both engineered tendons exhibited histology similar to that of natural tendon. Collagens became parallel throughout the tendon structure, and PGA fibers were completely degraded. Interestingly, dermal fibroblast and tenocyte engineered tendons did not express type III collagen at 26 weeks, which remained observable in normal pig skin and control group 2 tissue using polarized microscopy, suggesting a possible phenotype change of implanted dermal fibroblasts. Furthermore, both fibroblast and tenocyte engineered tendons shared similar tensile strength, about 75% of natural tendon strength. At 6 weeks in control group 2, neo-tissue was formed only at the peripheral area by host cells. A cord-like tissue was formed at weeks 14 and 26. However, the formed tissue was histologically disorganized and mechanically weaker than both cell-engineered tendons (p < 0.05). These results suggest that dermal fibroblasts may have the potential as seed cells for tendon engineering.