Gender‐specific in vivo measurement of the structural and mechanical properties of the human patellar tendon

Human patellar tendon stress (σ), strain (ε), stiffness (K), and tensile or Young's modulus (E), are determined in vivo through voluntary isometric contractions monitored with B‐mode ultrasonography. The limitations in previous studies are: (1) they have generally not accounted for the fact that the distal attachment of the patellar tendon (the tibial tuberosity) also displaces; thus, they have underestimated ε (and, hence, injury risk) while overestimating K; (2) no gender effect has been studied despite the fact that females are seen to have higher incidences of tendon‐related injuries. The current investigation therefore aimed to determine the gender specific values of σ, ε, K, and E of the patellar tendon while also accounting for distal displacement of the patellar tendon. Healthy young males (aged 23.1 ± 1.3 years, n = 10) and females (aged 21.3 ±0.9 years, n = 10) were tested. The maximal ε of the young males was ～5–10% higher than that reported in earlier literature. Average female versus male values for ε, σ, K, and E, taken at the same force level as the males for comparison purposes, were respectively 10.6 ± 1.0 versus 9.0 ± 1.0%, 36.9 ± 1.4 versus 28.9 ± 0.9 MPa, 1053 ± 108 versus 1652 ± 216 N · mm^?1, and 0.61 ± 0.08 versus 0.68 ± 0.10 GPa (p < 0.05). There are gender differences in tendon structural and mechanical properties. The current methodology may be useful in a clinical context where early prediction of injury risk and/or monitoring of reconstructed tendon needs to be an accurate, objective, and reliable method if optimal functionality is to be achieved. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1635–1642, 2007     

Functional tissue engineering for tendon repair: A multidisciplinary strategy using mesenchymal stem cells,bioscaffolds, and mechanical stimulation

Over the past 8 years, our group has been continuously improving tendon repair using a functional tissue engineering (FTE) paradigm. This paradigm was motivated by inconsistent clinical results after tendon repair and reconstruction, and the modest biomechanical improvements we observed after repair of rabbit central patellar tendon defects using mesenchymal stem cell‐gel‐suture constructs. Although possessing a significantly higher stiffness and failure force than for natural healing, these first generation constructs were quite weak compared to normal tendon. Fundamental to the new FTE paradigm was the need to determine in vivo forces to which the repair tissue might be exposed. We first recorded these force patterns in two normal tendon models and then compared these peak forces to those for repairs of central defects in the rabbit patellar tendon model (PT). Replacing the suture with end‐posts in culture and lowering the mesenchymal stem cell (MSC) concentration of these constructs resulted in failure forces greater than peak in vivo forces that were measured for all the studied activities. Augmenting the gel with a type I collagen sponge further increased repair stiffness and maximum force, and resulted in the repair tangent stiffness matching normal stiffness up to peak in vivo forces. Mechanically stimulating these constructs in bioreactors further enhanced repair biomechanics compared to normal. We are now optimizing components of the mechanical signal that is delivered in culture to further improve construct and repair outcome. Our contributions in the area of tendon functional tissue engineering have the potential to create functional load‐bearing repairs that will revolutionize surgical reconstruction after tendon and ligament injury. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1–9, 2008     

Non‐enzymatic glycation of chondrocyte‐seeded collagen gels for cartilage tissue engineering

Collagen glycated with ribose (250 mM) in solution (pre‐glycation) and as a gel (post‐glycation) was seeded with chondrocytes and the effects of glycation on chondrocyte matrix assembly in culture were determined. Pre‐glycation enhanced GAG accumulation significantly over controls at both 2 and 4 weeks (p < 0.05), although at both time points there were no statistical differences in cell number between pre‐glycated and control gels. The increased proteoglycan accumulation was shown to be in part due to significantly increased GAG retention by the pre‐glycated constructs (p < 0.05). Total collagen content in these pre‐glycated gels was also significantly higher than unglycated gels at 4 weeks (p < 0.05). With post‐glycation of collagen gels, chondrocyte number and GAG accumulation were all significantly lower than controls (p < 0.05). Post‐glycation also inhibited GAG retention by the constructs (p < 0.05). Given these results, pre‐glycation may be an improved processing method for collagen gels for tissue engineering techniques. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1434–1439, 2008     

Cross‐link stabilization does not affect the response of collagen molecules,fibrils, or tendons to tensile overload

We investigated whether immature allysine‐derived cross‐links provide mechanically labile linkages by exploring the effects of immature cross‐link stabilization at three levels of collagen hierarchy: damaged fibril morphology, whole tendon mechanics, and molecular stability. Tendons from the tails of young adult steers were either treated with sodium borohydride (NaBH₄) to stabilize labile cross‐links, exposed only to the buffer used during stabilization treatment, or maintained as untreated controls. One‐half of each tendon was then subjected to five cycles of subrupture overload. Morphologic changes to collagen fibrils resulting from overload were investigated using scanning electron microscopy, and changes in the hydrothermal stability of collagen molecules were assessed using hydrothermal isometric tension testing. NaBH₄ cross‐link stabilization did not affect the response of tendon collagen to tensile overload at any of the three levels of hierarchy studied. Cross‐link stabilization did not prevent the characteristic overload‐induced mode of fibril damage that we term discrete plasticity. Similarly, stabilization did not alter the mechanical response of whole tendons to overload, and did not prevent an overload‐induced thermal destabilization of collagen molecules. Our results indicate that hydrothermally labile cross‐links may not be as mechanically labile as was previously thought. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1907–1913, 2013     

Skeletal repair in rabbits using a novel biomimetic composite based on adipose‐derived stem cells encapsulated in collagen I gel with PLGA‐β‐TCP scaffold

In bone tissue engineering, the cell distribution mode in the scaffold may affect in vivo osteogenesis. Therefore, we fabricated a novel biomimetic construct based on a combination of rabbit adipose‐derived stem cells (rASCs) encapsulated in collagen I gel with a PLGA‐β‐TCP scaffold (rASCs‐COL/PLGA‐β‐TCP, group A), the combination of rASCs and PLGA‐β‐TCP (rASCs/PLGA‐β‐TCP, group B), the combination of collagen I gel and PLGA‐β‐TCP (COL/PLGA‐β‐TCP, group C), and PLGA‐β‐TCP scaffold (group D). The composites were implanted into a 15‐mm length critical‐sized segmental radial defect. The results were assessed by histology, radiographs, bone mineral density (BMD), and mechanical testing. After 24 weeks, the medullary cavity recanalized, bone was rebuilt, and molding finished, the bone contour remodeled smoothly and the scaffold degraded completely in group A. The BMDs and mechanical properties were similar to normal. However, the bone defect remained unrepaired in groups B, C, and D. Moreover, the scaffold degradation rate in group A was significantly higher than the other groups. Thus, enhanced in vivo osteogenesis of rASCs wrapped in collagen I gel combined with PLGA‐β‐TCP was achieved, and the bone defect was repaired. We hope this study provides new insights into ASCs‐based bone tissue engineering. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:252–257, 2010     

Effects of confinement on the mechanical properties of self‐assembled articular cartilage constructs in the direction orthogonal to the confinement surface

This study examined the effects of radial confinement and passive axial compression‐induced vertical confinement, on the biomechanical, biochemical, and histological properties of self‐assembled chondrocyte constructs. The self‐assembled constructs, engineered without the use of an exogenous scaffold, exhibited significant increases in stiffness in the direction orthogonal to that of the confinement surface. With radial confinement, the significantly increased aggregate modulus was accompanied by increased collagen organization in the direction perpendicular to the articular surface, with no change in collagen or glycosaminoglycan (GAG) content. Additionally, radial confinement was most beneficial when applied before 2 weeks. With passive axial compression, the significantly increased Young's modulus and ultimate tensile strength were accompanied by a significant increase in collagen production. This study is the first to demonstrate the beneficial effects of confinement on tissue engineered constructs in the direction orthogonal to that of the confinement surface. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:238–246, 2008     

Comparative Evaluation of the Book‐Type Acellular Bone Scaffold and Fibrocartilage Scaffold for Bone‐Tendon Healing

Bone‐tendon (B‐T) healing is a clinical challenge due to its limited regeneration capability. Fibrocartilage regeneration and bone formation at the healing site are two critical factors for B‐T healing. Promoting fibrocartilage regeneration and bone formation by tissue‐engineering may be a promising treatment strategy. In this study, we innovatively fabricated two kinds of acellular scaffolds from bone or fibrocartilage tissues, namely the book‐type the acellular bone scaffold (BABS) and the book‐type acellular fibrocartilage scaffold (BAFS). Histologically, the two scaffolds well preserved the native extracellular matrix (ECM) structure without cellular components. In vitro studies showed BABS is superior in osteogenic inducibility, while BAFS has good chondrogenic inducibility. To comparatively investigate the efficacy on B‐T healing, the BABS or BAFS were, respectively, implanted into a rabbit partial patellectomy model. Macroscopically, a regenerated bone‐tendon insertion (BTI) was bridging the residual patella and patellar‐tendon with no signs of infection and osteoarthritis. Radiologically, more new bone was formed at the healing interface in the BABS group as compared with the BAFS or control (CTL) groups (p < 0.05). Histologically, at postoperative week 16, histological scores were significantly better for regenerated fibrocartilage in the BAFS group or BABS group compared with the CTL group, but the BAFS group showed a significantly larger score than the BABS groups (p < 0.05). Biomechanical evaluation indicated a higher failure load and stiffness were shown in the BAFS group than those in the BABS or CTL groups at week 16 (p < 0.05). This study indicated that the BAFS is a more promising scaffold for B‐T healing in comparison with the BABS. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1709–1722, 2019     

Role of dexamethasone in the long‐term functional maturation of MSC‐laden hyaluronic acid hydrogels for cartilage tissue engineering

The purpose of study was to investigate the maturation of mesenchymal stem cells (MSC) laden in HA constructs with various combinations of chemically defined medium (CM) components and determine the impact of dexamethasone and serum on construct properties. Constructs were cultured in CM with the addition or withdrawal of media components or were transferred to serum containing media that partially represents an in vivo‐like condition where pro‐inflammatory signals are present. Constructs cultured in CM+ (CM with TGF‐β3) and DEX? (CM+ without dexamethasone) conditions produced robust matrix, while those in ITS/BSA/LA? (CM+ without ITS/BSA/LA) and Serum+ (10% FBS with TGF‐β3) produced little matrix. While construct properties in DEX? were greater than those in CM+ at 4 weeks, properties in CM+ and DEX? reversed by 8 weeks. While construct properties in DEX? were greater than those in CM+ at 4 weeks, the continued absence or removal of dexamethasone resulted in marked GAG loss by 8 weeks. Conversely, the continued presence or new addition of dexamethasone at 4 weeks further improved or maintained construct properties through 8 weeks. Finally, when constructs were converted to Serum (in the continued presence of TGF‐β3 with or without dexamethasone) after pre‐culture in CM+ for 4 weeks, GAG loss was attenuated with addition of dexamethasone. Interestingly, however, collagen content and type was not impacted. In conclusion, dexamethasone influences the functional maturation of MSC‐laden HA constructs, and may help to maintain properties during long‐term culture or with in vivo translation by repressing pro‐inflammatory signals. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1717–1727, 2018.

     

Repair of large osteochondral defects in rabbits using porous hydroxyapatite/collagen (HAp/Col) and fibroblast growth factor‐2 (FGF‐2)

Articular cartilage has a limited capacity for self‐renewal. This article reports the development of a porous hydroxyapatite/collagen (HAp/Col) scaffold as a bone void filler and a vehicle for drug administration. The scaffold consists of HAp nanocrystals and type I atelocollagen. The purpose of this study was to investigate the efficacy of porous HAp/Col impregnated with FGF‐2 to repair large osteochondral defects in a rabbit model. Ninety‐six cylindrical osteochondral defects 5 mm in diameter and 5 mm in depth were created in the femoral trochlear groove of the right knee. Animals were assigned to one of four treatment groups: porous HAp/Col impregnated with 50 µl of FGF‐2 at a concentration of 10 or 100 µg/ml (FGF10 or FGF100 group); porous HAp/Col with 50 µl of PBS (HAp/Col group); and no implantation (defect group). The defect areas were examined grossly and histologically. Subchondral bone regeneration was quantified 3, 6, 12, and 24 weeks after surgery. Abundant bone formation was observed in the HAp/Col implanted groups as compared to the defect group. The FGF10 group displayed not only the most abundant bone regeneration but also the most satisfactory cartilage regeneration, with cartilage presenting a hyaline‐like appearance. These findings suggest that porous HAp/Col with FGF‐2 augments the cartilage repair process. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:677–686, 2010     

Mechanical loading increased BMP‐2 expression which promoted osteogenic differentiation of tendon‐derived stem cells

This study aimed to investigate the effect of repetitive tensile loading on the expression of BMP‐2 and the effect of BMP‐2 on the osteogenic differentiation of tendon‐derived stem cells (TDSCs) in vitro. Repetitive stretching was applied to TDSCs isolated from rat patellar tendon at 0%, 4%, and 8%, 0.5 Hz. The expression of BMP‐2 was detected by Western blotting and qPCR. To study the osteogenic effects of BMP‐2 on TDSCs, BMP‐2 was added to the TDSC monolayer for the detection of ALP activity and calcium nodule formation in a separate experiment. TDSCs adhered, proliferated, and aligned along the direction of externally applied tensile force while they were randomly oriented in the control group. Western blotting showed increased expression of BMP‐2 in 4% and 8% stretching groups but not in the control group. Up‐regulation of BMP‐2 mRNA was also observed in the 4% stretching group. BMP‐2 increased the osteogenic differentiation of TDSCs as indicated by higher ALP cytochemical staining, ALP activity, and calcium nodule formation. Repetitive tensile loading increased the expression of BMP‐2 and addition of BMP‐2 enhanced osteogenic differentiation of TDSCs. Activation of BMP‐2 expression in TDSCs during tendon overuse might provide a possible explanation of ectopic calcification in calcifying tendinopathy. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:390–396, 2011     

Effective control of oozing from remnant tissue after partial nephrectomy,using fibrin tissue‐adhesive collagen fleece

A 76‐year‐old man had complained of vomiting and diarrhea. A low echoic mass shown by abdominal ultrasonography with a clear margin in the right kidney was revealed. Digital subtraction angiography revealed a mass containing granular vascularity that was 18 mm in diameter and was protruding from the dorsal aspect of the kidney. He was diagnosed as having an encapsulated right renal cell carcinoma (T1N0M0, stage I). Partial nephrectomy was conducted as his renal function was poor. This was indicated by a preoperative creatinine clearance of 44.4 mL/min. After the resection of the tumour, the haemorrhage from the renal parenchyma did not stop by suturing. To control the oozing from the resection surface by electrical coagulation, liquid fibrin glue and powdered fibrin glue was used, however it was not successful. Thus, we decided to use fibrin tissue‐adhesive collagen fleece (FTCF, TachoComb; Nycomed Pharma, Linz, Austria). The FTCF was placed on the resection surface and a pressure was applied to it for 5 min; oozing stopped completely. The use of FTCF contributed to the preservation renal function as there was no need to keep the seams as compared with suturing. The usefulness of FTCF in the partial nephrectomy is discussed in the present case report.      

Isolation and characterization of turkey bone marrow‐derived mesenchymal stem cells

Flexor tendon injury is often associated with suboptimal outcomes and results in substantial digit dysfunction. Stem cells have been isolated from several experimental animals for the growing interest and needs of utilizing cell‐based therapies. Recently, turkey has been developed as a new large animal model for flexor tendon research. In the present study, we reported the isolation and characterization of bone marrow‐derived mesenchymal stem cells (BMSCs) from 8‐ to 12‐month‐old heritage‐breed turkeys. The isolated cells demonstrated fibroblast‐like morphology, clonogenic capacity, and high proliferation rate. These cells were positive for surface antigens CD90, CD105, and CD44, but were negative for CD45. The multipotency of turkey BMSCs was determined by differentiating cells into osteogenic, adipogenic, chondrogenic, and tenogenic lineages. There was upregulated gene expression of tenogenic markers, including mohawk, tenomodulin, and EGR1 as well as increased collagen synthesis in BMP12 induced cells. The successful isolation and verification of bone marrow‐derived MSCs from turkey would provide opportunities of studying cell‐based therapies and developing new treatments for tendon injuries using this novel preclinical large animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1419–1428, 2019.     

Tissue‐engineered tendon constructs for rotator cuff repair in sheep

Current rotator cuff repair commonly involves the use of single or double row suture techniques, and despite successful outcomes, failure rates continue to range from 20 to 95%. Failure to regenerate native biomechanical properties at the enthesis is thought to contribute to failure rates. Thus, the need for technologies that improve structural healing of the enthesis after rotator cuff repair is imperative. To address this issue, our lab has previously demonstrated enthesis regeneration using a tissue‐engineered graft approach in a sheep anterior cruciate ligament (ACL) repair model. We hypothesized that our tissue‐engineered graft designed for ACL repair also will be effective in rotator cuff repair. The goal of this study was to test the efficacy of our Engineered Tissue Graft for Rotator Cuff (ETG‐RC) in a rotator cuff tear model in sheep and compare this novel graft technology to the commonly used double row suture repair technique. Following a 6‐month recovery, the grafted and contralateral shoulders were removed, imaged using X‐ray, and tested biomechanically. Additionally, the infraspinatus muscle, myotendinous junction, enthesis, and humeral head were preserved for histological analysis of muscle, tendon, and enthesis structure. Our results showed that our ETC‐RCs reached 31% of the native tendon tangent modulus, which was a modest, non‐significant, 11% increase over that of the suture‐only repairs. However, the histological analysis showed the regeneration of a native‐like enthesis in the ETG‐RC‐repaired animals. This advanced structural healing may improve over longer times and may diminish recurrence rates of rotator cuff tears and lead to better clinical outcomes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:289–299, 2018.     

Construction of tissue‐engineered corpus cavernosum with muscle‐derived stem cells and transplantation in vivo

Study Type – Therapy (case series)
Level of Evidence 4 What’s known on the subject? and What does the study add? The tissue‐engineered research of corpus cavernosum has been studied, but an ideal method was not carried out. In the study, muscle‐derived stem cells were used as seeding cells to construct tissue‐engineered corpus cavernosums. The result demonstrated MDSCs could be seeded on three‐dimensional scaffolds of acellular corporal collagen matrices and developed into tissues similar to native corpus cavernosum in vivo.

OBJECTIVE

• ? To investigate the feasibility of tissue‐engineered corpus cavernosum (TECC) with muscle‐derived stem cells (MDSCs) as seed cells and determine the growth potential in vivo.

MATERIALS AND METHODS

• ? Acellular corporal collagen matrices (ACCMs) were obtained from adult rabbit penis by a cell removal procedure. MDSCs were separated and purified using a digestion method and Preplate technique, then seeded on ACCMs at a concentration of 30 × 10⁶ cells/mL to construct TECCs. After 5 days of culture, seeded ACCMs were implanted with albuginea of rabbits. The implants were retrieved at 2, 4 and 6 months after implantation.
• ? Histochemistry, immunohistochemisry and scanning electron microscopy were performed to analyse the morphological characteristics of the TECCs.

RESULTS

• ? The decellularization process successfully extracted all cellular components while preserving the original collagen fibres.
• ? Histological analyses of the explants at all time points in the experimental group had more cells and better arranged growth than the control group. α‐Smooth muscle actin and endothelial nitric oxide synthase‐positive cells were more prevalent in the experimental group.

CONCLUSION

• ? Our study showed that MDSCs can be seeded on three‐dimensional ACCM scaffolds and develop tissues that are similar to native normal corpus cavernosum.

     

Smad8/BMP2‐engineered mesenchymal stem cells induce accelerated recovery of the biomechanical properties of the achilles tendon

Tendon tissue regeneration is an important goal for orthopedic medicine. We hypothesized that implantation of Smad8/BMP2‐engineered MSCs in a full‐thickness defect of the Achilles tendon (AT) would induce regeneration of tissue with improved biomechanical properties. A 2 mm defect was created in the distal region of murine ATs. The injured tendons were then sutured together or given implants of genetically engineered MSCs (GE group), non‐engineered MSCs (CH3 group), or fibrin gel containing no cells (FG group). Three weeks later the mice were killed, and their healing tendons were excised and processed for histological or biomechanical analysis. A biomechanical analysis showed that tendons that received implants of genetically engineered MSCs had the highest effective stiffness (>70% greater than natural healing, p < 0.001) and elastic modulus. There were no significant differences in either ultimate load or maximum stress among the treatment groups. Histological analysis revealed a tendon‐like structure with elongated cells mainly in the GE group. ATs that had been implanted with Smad8/BMP2‐engineered stem cells displayed a better material distribution and functional recovery than control groups. While additional study is required to determine long‐term effects of GE MSCs on tendon healing, we conclude that genetically engineered MSCs may be a promising therapeutic tool for accelerating short‐term functional recovery in the treatment of tendon injuries. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1932–1939, 2012     

The relationships among spatiotemporal collagen gene expression,histology, and biomechanics following full‐length injury in the murine patellar tendon

Tendon injuries are major orthopedic problems that worsen as the population ages. Type‐I (Col1) and type‐II (Col2) collagens play important roles in tendon midsubstance and tendon‐to‐bone insertion healing, respectively. Using double transgenic mice, this study aims to spatiotemporally monitor Col1 and Col2 gene expression, histology, and biomechanics up to 8 weeks following a full‐length patellar tendon injury. Gene expression and histology were analyzed weekly for up to 5 weeks while mechanical properties were measured at 1, 2, 5, and 8 weeks. At week 1, the healing region displayed loose granulation tissue with little Col1 expression. Col1 expression peaked at 2 weeks, but the ECM was highly disorganized and hypercellular. By 3 weeks, Col1 expression had reduced and by 5 weeks, the ECM was generally aligned along the tendon axis. Col2 expression was not seen in the healing midsubstance or insertion at any time point. The biomechanics of the healing tissue was inadequate at all time points, achieving ultimate loads and stiffnesses of 48% and 63% of normal values by 8 weeks. Future studies will further characterize the cells within the healing midsubstance and insertion using tenogenic markers and compare these results to those of tendon cells during normal development. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:28–36, 2012     

Increasing platelet concentration in platelet‐rich plasma inhibits anterior cruciate ligament cell function in three‐dimensional culture

Tissue engineering is one new strategy being developed to treat ACL ruptures. One such approach is bio‐enhanced ACL repair, where a suture repair is supplemented with a bio‐active scaffold containing platelets. However, the optimal concentration of platelets to stimulate ACL healing is not known. We hypothesized that increasing platelet concentrations in the scaffold would enhance critical cell behaviors. Porcine ACL fibroblasts were obtained from explant culture and suspended in platelet poor plasma (PPP), 1× platelet‐rich plasma (PRP), 3× PRP, 5× PRP, or phosphate buffered saline (PBS). The cell suspensions were cultured in a 3D collagen scaffold. Cellular metabolism (MTT assay), apoptosis (TUNEL assay), and gene expression for type I and type III collagen were measured. 1× PRP significantly outperformed 5× PRP in all parameters studied: Type I and III collagen gene expression, apoptosis prevention, and cell metabolism stimulation. ACL fibroblasts cultured with 1× PRP had the highest type I and type III collagen gene expression. 1× PRP and PPP groups had the highest cell metabolism and lowest apoptosis rates. Concentration of platelets had significant effects on the behavior of ACL fibroblasts; thus, it is an important parameter that should be specified in clinical or basic science studies. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:291–295, 2014.