Extracellular matrix expression of human tenocytes in three‐dimensional air–liquid and PLGA cultures compared with tendon tissue: Implications for tendon tissue engineering

Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell–cell and cell–matrix contacts within three‐dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic‐co‐glycolic‐acid] (PLGA)‐scaffolds and high‐density cultures. Neotissue formation was examined by hematoxyline–eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon‐like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4‐week‐old 3D cultures, whereas type III collagen was only elevated in high‐density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high‐density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon‐like tissue. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1170–1177, 2010     

Mechanism of mast cell adhesion to human tenocytes in vitro

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell‐fibroblast co‐culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte‐conditioned media; the tenocyte‐derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte‐derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte‐derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:9–16, 2015.

     

腱细胞与人工材料体外联合培养的形态学观察

目的：为了探索肌腱细胞与人工材料是否有可能复合形成新型有活性的人工肌腱，特设计了本项研究。方法：取第４代腱细胞，复苏后分别加入碳纤维编织带、涤纶编织带及几丁质编织带联合培养，在倒置显微镜及透射电镜下观察形态学改变及细胞与材料的相容性。结果：腱细胞与碳纤维有良好的相容性。腱细胞沿碳纤维生长、繁殖，并合成胶原。腱细胞与涤纶的相容性较差，在几丁质上无细胞附着。结论：腱细胞与碳纤维联合培养后，保持了腱细胞的形态特征，与碳纤维的相容性最好。有可能成为一种新型有活性的人工肌腱材料     

Characterization of a purified exosome product and its effects on canine flexor tenocyte biology

Flexor tendon injuries and tendinopathy are very common but remain challenging in clinical treatment. Exosomes-based cell-free therapy appears to be a promising strategy for tendon healing, while limited studies have evaluated its impacts on tenocyte biology. The objective of this study was to characterize a novel purified exosome product (PEP) derived from plasma, as well as to explore its cellular effects on canine tenocyte biology. The transmission electron microscope revealed that exosomes of PEP present cup-shaped structures with the diameters ranged from 80 to 141 nm, and the NanoSight report presented that their size mainly concentrated around 100 nm. The enzyme-linked immunosorbent assay kits analysis showed that PEP was positive for CD63 and AChE expression, and the cellular uptake of exosomes internalized into tenocyte cytoplasm was observed. The cell growth assays displayed that tenocyte proliferation ability was enhanced by PEP solution in a dose-dependent manner. Tenogenic phenotype was preserved as is evident by that tendon-related genes expression (SCX, COL1A, COL3A1, TNMD, DCN, and MKX) were expressed insistently in a high level, while tenocytes were treated with 5% PEP solution. Furthermore, migration capability was maintained and total collagen deposition was increased. More interesting, dexamethasone-induced cellular apoptosis was attenuated during the incubation of tenocytes with a 5% PEP solution. These findings will provide the basic understandings about the PEP, and support the potential use of this biological strategy for tendon healing.     

生物衍生材料构建组织工程肌腱体内植入的实验研究

目的 探讨用同种异体肌腱细胞与生物衍生肌腱材料体外联合培养后植入体内，构建组织工程肌腱的可行性。方法 选用四川锦猴15只，手术造成纤维鞘管内屈指深肌腱2．5cm缺损后，分三组。A组：生物衍生肌腱材料构建的组织工程肌腱移植组；B组：单纯生物衍生肌腱材料移植组；C组：自体肌腱移植组。术后1、2、3、6和12周分期观察植入物的大体形态、组织学和超微结构，BrdU标记表达。结果 A组植入体内后肌腱细胞能继续增殖，细胞形态随着时间增加而逐渐趋于正常，形成的肌腱呈白色且有光泽、致密，组织学可见胶原纤维排列较为规则，12周肌腱细胞仍成活并分泌胶原，BrdU表达为阳性；B组植入体内3周后材料逐渐变细，12周后材料被逐渐降解吸收出现中断；C组植入体内2周后桥接部有纤维连接，排列较为规则，肌腱愈合。A组分别于3、6、12周进行扫描电镜观察可见肌腱细胞排列均匀，胶原纤维相互连接形成网状，主体趋势与肌腱走行方向一致；透射电镜下可见细胞核仁清晰，细胞器丰富。随时间的增加A、C组与B组的差异明显增大。结论 同种异体肌腱细胞与生物衍生肌腱材料复合构建的组织工程肌腱，植入免疫功能正常的动物体内能够再生出肌腱样组织，植入的肌腱细胞具有生命力，新生的肌腱组织在大体、组织学方面与正常肌腱相似。     

Low-level laser irradiation stimulates tenocyte proliferation in association with increased NO synthesis and upregulation of PCNA and cyclins

Low-level laser therapy is commonly used to treat tendinopathy or tendon injury. Tendon healing requires tenocyte migration to the repair site, followed by proliferation and synthesis of the extracellular matrix. There are few evidence to elucidate that low-level laser promote tenocyte proliferation. This study was designed to determine the effect of laser on tenocyte proliferation. Furthermore, the association of this effect with secretion of nitric oxide (NO) and the expressions of proliferating cell nuclear antigen (PCNA) and cyclins D1, E, A, and B1 was investigated. Tenocytes intrinsic to rat Achilles tendon were treated with low-level laser (660 nm). Tenocyte proliferation was evaluated by MTT assay and immunocytochemistry with Ki-67 stain. NO in the conditioned medium was measured by ELISA. Western blot analysis was used to evaluate the protein expressions of PCNA and cyclins D1, E, A, and B1. The results revealed that tenocytes proliferation was enhanced dose dependently by laser. NO secretion was increased after laser treatment. PCNA and cyclins E, A, and B1 were upregulated by laser. In conclusion, low-level laser irradiation stimulates tenocyte proliferation in a process that is mediated by upregulation of NO, PCNA, and cyclins E, A, and B1.     

Triamcinolone suppresses human tenocyte cellular activity and collagen synthesis

Glucocorticoid injection is widely used in tendon disorders. Despite previous studies on the histologic and biomechanical changes in tendons after glucocorticoid injections, the role of glucocorticoid in tendon rupture still is controversial. It was hypothesized that glucocorticoid has a direct deleterious effect on human tenocytes, suppressing its cellular activity and collagen production. Primary cultures of human tenocytes were obtained from explants of healthy patellar tendon harvested during anterior cruciate ligament reconstructions. The effects on cell viability and cell proliferation were measured by [3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and 5-bromo-deoxyuridine incorporations. The effect on collagen synthesis was measured by H-proline incorporation assay. Triamcinolone acetonide at 10 to 10 mol/L decreased human tenocyte viability to 45% to 88% of control in a dose-dependent manner. Cell proliferation was suppressed to 87% +/- 8% at all doses. Treatment with 1 micromol/L triamcinolone acetonide reduced the amount of collagen synthesis as measured by H-proline incorporation from 40 +/- 2 cpm/1000 cells to 27 +/- 4 cpm/1000 cells. The suppressed human tenocyte cellular activity and reduced collagen production may lead to disturbed tendon structure and predispose the tendon to subsequent spontaneous rupture.     

Proliferation and differentiation of human tenocytes in response to platelet rich plasma: an in vitro and in vivo study

Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet-rich cellular component which is enriched with a number of growth factors. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for various applications, for example, tendon regeneration but with limited success in clinical trials. The main objective of the current study was to determine whether activated PRP [i.e., platelet rich plasma-clot release (PRCR)] could be used to induce the proliferation and collagen synthesis in human tenocyte in vitro. The advantage of using PRCR is that the platelet-derived bioactive factors are more concentrated and could initiate a more rapid and accelerated healing response than PRP. Our results demonstrated that 10% PRCR treatment accelerated the extent of cell proliferation and collagen production by human tenocytes in vitro. The expression of specific tenocyte markers were similar to conventional fetal bovine serum (FBS)-treated tenocytes implanted in mice within 14 days of implantation in diffusion chambers. Moreover, relatively more collagen fibrils were evident in PRCR-treated tenocytes in vivo as compared to 10% FBS-treated cells. Overall, our feasibility study has indicated that PRCR can induce human tenocyte proliferation and collagen synthesis which could be implemented for future tendon regeneration in reconstructive surgeries.     

The impact of cell culture equipment on energy loss

Light energy of discrete wavelengths supplied via lasers and broadband intense pulsed light have been used therapeutically for many years. In vitro models complement clinical studies, especially for the elucidation of underlying mechanisms of action. Clarification that light energy reaches the cells is necessary when developing protocols for the treatment of cells using in vitro models. Few studies report on energy loss in cell culture equipment. The ability of energy from light with therapeutic potential to reach cells in culture needs to be determined; this includes determining the proportion of light energy lost within standard cell culture media and cell culture vessels. The energy absorption of cell culture media, with/without the pH indicator dye phenol red, and the loss of energy within different plastics and glassware used typically for in vitro cell culture were investigated using intense pulsed light and a yellow pulsed dye laser. Media containing phenol red have a distinctive absorption peak (560 nm) absent in phenol red-free media and restored by the addition of phenol red. For both light sources, energy loss was lowest in standard polystyrene tissue culture flasks or multi-well plates and highest in polypropylene vessels or glass tubes. The effects of phenol red-free media on the absorption of energy varied with the light source used. Phenol red-free media are the media of choice; polystyrene vessels with flat surfaces such as culture flasks or multi-well plates should be used in preference to polypropylene or glass vessels.     

Glucocorticoids inhibit tenocyte proliferation and Tendon progenitor cell recruitment.

Corticosteroid injection is commonly used to treat tendon injuries but is often associated with tendon rupture and impaired tendon healing. The effects of dexamethasone on tenocytes have been studied in vitro but only using high concentrations of dexamethasone in monolayer cultures of tenocytes over short periods of time. We have therefore investigated the effects of physiological and pharmacological concentrations of dexamethasone on monolayer cultures of tenocytes over extended time periods. We have also used fibroblastic-colony forming unit cultures to examine the effects of dexamethasone on a progenitor cell population located in tendons. Culturing tenocytes in the presence of dexamethasone for a period of 24 days resulted in a concentration-related decrease in cell number and collagen synthesis as compared to control cultures. This effect was time dependent with cell number in both dexamethasone-treated and control cultures leveling off after 14 days with the control cultures reaching higher cell densities. In contrast in control cultures, collagen accumulation continued to increase until week 4, whereas in the presence of dexamethasone, this tended to level off after 14 days. To study the role of progenitor cell recruitment, the effects of dexamethasone were investigated using the fibroblastic-colony forming unit assay. Treatment with dexamethasone at concentrations of 0.1 nM to 10 microM leads to a progressive reduction in mean colony size as compared to control cultures. Colony number remained constant at concentrations below 10 nM but fell progressively at concentrations above this. In conclusion, dexamethasone reduces both cell number and collagen synthesis in tenocyte cultures in a concentration-dependent manner by both direct effects on tenocyte proliferation and collagen accumulation, and also by modulating the recruitment of tendon progenitor cells.     

A comparison of lidocaine, ropivacaine and dexamethasone toxicity on bovine tenocytes in culture

Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic. Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation.     

Role of pulsed electromagnetic fields (PEMF) on tenocytes and myoblasts—potential application for treating rotator cuff tears

The post‐surgery integrity of the tendons and muscle quality are the two major factors in success of rotator cuff (RC) repair. Though surgical techniques for rotator cuff repair have significantly improved in the past two decades, there are no effective treatments to improve tendon‐to‐bone healing and muscle quality after repair at this point in time. Pulsed electromagnetic fields (PEMF) have previously been used for promoting fracture healing. Previous studies have shown that PEMF has a positive role in promoting osteoblast precursors proliferation and differentiation. However, PEMFs effect on tenocytes and muscle cells has not been determined fully yet. The purpose of this study is to define the role of a commercially available PEMF on tenocytes and myoblasts growth and differentiation in vitro. Human rotator cuff tenocytes and C2C12 murine myoblasts were cultured and treated with PEMF for 2 weeks under regular and inflammatory conditions. Our results showed that 2 weeks treatment of PEMF enhanced gene expressions of growth factors in human rotator cuff tenocytes under inflammatory conditions. PEMF significantly enhanced C2C12 myotube formation under normal and inflammatory conditions. Results from this study suggest that PEMF has a positive role in promoting tenocyte gene expression and myoblast differentiation. Therefore, PEMF may potentially serve as a non‐operative treatment to improve clinical incomes rotator cuff tendon repairs. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:956–964, 2017.

     

Prolotherapy Induces an Inflammatory Response in Human Tenocytes In Vitro

Background

Proliferative therapy, or prolotherapy, is a controversial treatment method for many connective tissue injuries and disorders. It involves the injection of a proliferant, or irritant solution, into the site of injury, which causes small-scale cell death. This therapeutic trauma is theorized to initiate the body’s wound-healing cascade, perhaps leading to tissue repair. The immediate effects of many of these proliferants are poorly characterized, as are the cellular responses to them; here, we sought to evaluate the immediate effects of two common proliferants (dextrose and P2G, a combination of phenol, glucose, and glycerin) on the cellular response of human tenocytes, and begin to explicate the mechanisms with which each proliferant functions.

Questions/purposes

We asked: What are the effects of treating cultured tenocytes with proliferative treatment agents on their (1) cellular metabolic activity, (2) RNA expression, (3) protein secretion, and (4) cell migration?

Methods

Using human hamstring and Achilles tendon cells, we attempted to answer our research questions. We used a colorimetric metabolic assay to assess the effect of dextrose and P2G proliferant treatment on cell mitochondrial activity compared with nontreated tenocytes. Next, using quantitative PCR, ELISA, and a reporter cell line, we assessed the expression of several key markers involved in tendon development and inflammation. In addition, we used a scratch wound-healing assay to evaluate the effect of proliferant treatment on tenocyte migration.

Results

Results showed that exposure to both solutions led to decreased metabolic activity of tenocytes, with P2G having the more pronounced effect (75% ± 7% versus 95% ± 7% of untreated control cell metabolic levels) (ANOVA; p < 0.01; mean difference, 0.202; 95% CI, 0.052–0.35). Next, gene expression analysis confirmed that treatment led to the upregulation of key proinflammatory markers including interleukin-8 and cyclooxygenase-2 and downregulation of the matrix marker collagen type I. Furthermore, using a reporter cell line for transforming growth factor-β (TGF-β), a prominent antiinflammatory marker, we showed that treatments led to decreased TGF-β bioactivity. Analysis of soluble proteins using ELISA revealed elevated levels of soluble prostaglandin E2 (PGE2), a prominent inducer of inflammation. Finally, both solutions led to decreased cellular migration in the tenocytes.

Conclusions

Taken together, these results suggest that prolotherapy, more so with P2G, may work by decreasing cellular function and eliciting an inflammatory response in tenocytes. Additional studies are needed to confirm the cellular signaling mechanisms involved and the resulting immediate response in vivo.

Clinical Relevance

If these preliminary in vitro findings can be confirmed in an in vivo model, they may provide clues for a possible cellular mechanism of a common alternative treatment method currently used for certain soft tissue injuries.

     

Coordinate Regulation of IL-1β and MMP-13 in Rat Tendons Following Subrupture Fatigue Damage

Mechanical overloading is a major causative factor of tendinopathy; however, its underlying mechanisms are unclear. We hypothesized mechanical overloading would damage tendons and alter genes associated with tendinopathy in a load-dependent manner. To test this hypothesis, we fatigue loaded rat patellar tendons in vivo and measured expression of the matrix-degrading enzyme MMP-13 and the inflammatory cytokine IL-1β. We also examined these responses in cultured tenocytes exposed to intermittent hydrostatic pressure in vitro. Additionally, we hypothesized load-induced changes in tenocyte MMP-13 expression would be dependent on expression of IL-1β. In vivo fatigue loading at 1.7% strain caused overt microstructural damage and upregulated expression of MMP-13 and IL-1β, while 0.6% strain produced only minor changes in matrix microstructure and downregulated expression of both MMP-13 and IL-1β. Loading of cultured tenocytes at 2.5 and 7.5 MPa produced comparable changes in expression to those of in vivo tendon loading. Blocking IL-1β expression with siRNA suppressed load-induced both MMP-13 mRNA expression and activity. The data suggest fatigue loading alters expression of MMP-13 and IL-1β in tendons in vivo and tenocytes in vitro in a load-dependent manner. The data also suggest MMP-13 is regulated by both IL-1β-dependent and IL-1β-independent pathways. One or more of the authors (HBS, MBS, ELF) has received funding from the National Institutes of Health. This study was supported by grants from the Aircast Foundation and NIH Grants AR41210, AR52743, and AR50968. Each author certifies that his or her institution has approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.     

Biologic behavior of an in vitro hydrated collagen gel-human tenocyte tendon model

An in vitro human tenocyte-collagen gel model was developed to study tenocyte-mediated Type I collagen fibril reorganization, proliferation, and Type I collagen gene expression. Human tenocytes, obtained from extrasynovial forearm flexor tendons from children 5 to 10 years of age were cultured on plastic or in a cylinder of hydrated Type I collagen gel. Collagen solution was seeded with human tenocytes at 5 x 10(5) cells/mL and gelled in cylinder molds; gel cylinders without human tenocytes served as controls. Gel cylinders were pinned to troughs to create noncompliance. The gel cylinders were analyzed for collagen birefringence and cell shape at 7 and 21 days and for proliferation and gene expression for Type I collagen at 7 days. Under conditions of noncompliance, human tenocytes reoriented Type I collagen into longitudinal bundles resembling the parallel organization of collagen in native tendons. Tenocyte shape became fusiform between the collagen bundles which mimics the morphologic features of a tenocyte in vivo. The structural changes in the tenocytes and matrix are accompanied by downregulation of human tenocyte proliferation and Type I collagen gene expression. When released from the gel cylinder and grown again on plastic, human tenocytes resume proliferation and Type I collagen gene expression. The human tenocytes in this in vitro gel cylinder model system control fibril reorganization and proliferation, resembling their behavior during the development and repair of native tendons.     

深低温冷冻肌腱细胞活性的研究

目的研究深低温冷冻方法对肌腱细胞活性的影响,比较程序性降温和普通深低温冷冻法对腱细胞活性的影响.方法纯种SD大鼠24只（出生21 d）,随机分为3组,取双侧跟腱.新鲜肌腱对照组（A）,常规深低温冷冻组（B）,程序性降温深低温冷冻组（C）.采用相同的方法对3组肌腱细胞进行细胞培养.相差显微镜观察原代和传代后细胞的生长,绘制细胞的生长曲线,考察细胞的活性;对细胞进行成纤维细胞染色、胶原染色和对细胞进行形态观察（扫描电镜）;水解法定量分析细胞培养基中羟脯氨酸浓度的变化,检测细胞合成胶原的能力.结果原代细胞培养时A组细胞的生长速度快于B组和C组（P＜0.01）,C组细胞的生长速度快于B组（P＜0.01）,这种生长速度的差异在细胞传代后消失.细胞的形态学和组织学符合成纤维细胞形态.3组细胞培养基中羟脯氨酸浓度变化的差异无统计意义（P＞0.05）.结论经深低温冷冻处理的肌腱中仍存在具有活性的腱细胞,但数量显著少于新鲜肌腱中活细胞的数量.应用计算机控制程序性慢速降温方法处理的肌腱其活细胞的数量有所提高,但仍低于新鲜肌腱中活细胞的数量.     

VGluT2 expression in painful Achilles and patellar tendinosis: Evidence of local glutamate release by tenocytes

The pathogenesis of chronic tendinopathy is unclear. We have previously measured high intratendinous levels of glutamate in patients with tendinosis, suggesting potential roles of glutamate in the modulation of pain, vascular function, and degenerative changes including apoptosis of tenocytes. However, the origin of free glutamate found in tendon tissue is completely unknown. Surgical biopsies of pain‐free normal tendons and tendinosis tendons (Achilles and patellar) were examined immunohistochemically using antibodies against vesicular glutamate transporters (VGluT1 and VGluT2), as indirect markers of glutamate release. In situ hybridization for VGluT2 mRNA was also conducted. Specific immunoreactions for VGluT2, but not VGluT1, could be consistently detected in tenocytes. However, there were interindividual variations in the levels of immunoreactivity. The level of immunoreaction for VGluT2 was higher in tendinosis tendons compared to normal tendons (p < 0.05). In situ hybridization of VGluT2 demonstrated that mRNA was localized in a similar pattern as the protein, with marked expression by certain tenocytes, particularly those showing abnormal appearances. Reactivity for VGluT1 and ‐2 was absent from nerves and vessel structures in both normal and painful tendons. The current data demonstrate that tenocytes may be involved in the regulation of extracellular glutamate levels in tendons. Specifically, the observations suggest that free glutamate may be locally produced and released by tenocytes, rather than by peripheral neurons. Excessive free glutamate is expected to impact a variety of autocrine and paracrine functions important in the development of tendinosis, including tenocyte proliferation and apoptosis, extracellular matrix metabolism, nociception, and blood flow. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:685–692, 2008