Glutamate receptors in tendinopathic patients

Tendinopathy, pain, and degeneration, may be related to the up‐regulation of substance P (SP) and its activation of glutamate receptors. We hypothesized that the pathogenesis of tendinopathy involves N‐methyl‐D ‐aspartate receptor type 1 (NMDAR1) activation (phosphorylated NMDAR1; phospho‐NMDAR1) co‐existing with SP. Moreover, we examined the presence of metabotropic receptors that increase (mGluR1 and mGluR5) or decrease (mGluR6 and mGluR7) NMDAR1 excitability. Biopsies from patients with patellar tendinopathy (n = 10) and from controls (n = 8) were immunohistochemically analyzed according to the occurrence and tissue distribution of NMDAR1, phosho‐NMDAR1, mGluR (1, 5–7), and SP. The biopsies were immunohistochemically single‐ and double‐stained and semi‐quantitatively assessed. Tendinopathic biopsies exhibited increased occurrence of NMDAR1, phospho‐NMDAR1, SP, and mGluR5, while mGluR6–7 were not increased and mGluR1 was not found. The occurrence of NMDAR1 and SP correlated in tendinopathy (r² = 0.54, p = 0.03), but not in the controls (r² = 0.11, p = 0.4). Co‐localization of SP and phospho‐NMDAR1 within the tendon proper was only found in tendinopathy, localized on hypertrophic tenocytes, blood vessels, and penetrating free‐nerve fibres. Up‐regulation and activation of the glutamate receptor, phospho‐NMDAR1, suggests a role in the pathophysiology of tendinopathy. Increased NMDAR1 excitability may be related to increased SP and mGluR5. The unique co‐existence of SP and phospho‐NMDAR1 in tendinopathy presumably reflects a tissue proliferative and nociceptive role. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1447–1452, 2012     

Expression of chondro‐osteogenic BMPs in ossified failed tendon healing model of tendinopathy

Chondrocytes phenotype/markers were expressed in clinical samples of tendinopathy and calcifying tendinopathy. This study examined the spatial‐temporal expression of chondro‐osteogenic Bone Morphogenetic Proteins (BMPs), which might contribute to ectopic chondro‐osteogenesis and failed healing process in tendinopathy. Collagenase was injected into patellar tendon of rats to induce ossified failed tendon healing. At week 2, 4, 8, 12, and 16, the patella tendon was harvested for immunohistochemical staining and analysis of BMP‐2/4/7. BMP‐4/7 showed similar expression patterns, which was different from BMP‐2. The expression of BMP‐2 in the tendon matrix increased at week 2 and was reduced to nearly undetectable level afterwards except at the chondro‐ossification sites. However, the expression of BMP‐4/7 in the healing tendon fibroblast‐like cells and matrix increased at week 2, reduced at week 4 and 8 and increased again at week 12 and 16, consistent with transient healing at week 8 in this animal model. There was increasing strong expression of BMP‐4/7 in the chondrocyte‐like cells in the un‐ossified and ossified areas from week 8–16. BMP‐4/7, besides BMP‐2, might also contribute to ectopic chondro‐osteogenesis and failed healing in tendon injuries. BMP‐4/7, but not BMP‐2, might be involved in regulating late events in ossified failed tendon healing. © 2010 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:816–821     

Radix Dipsaci does not improve tendon healing in a rat model of patellar tendon donor site injury

Objective: To explore whether Radix Dipsaci (RD) exhibits beneficial effects on tendon healing. Methods: An attempt was made to explore the in vitro effects of a hot water extract of RD on gene expression of procollagen Type I (COL1A1), procollagen Type III (COL3A1) and decorin in cultured tendon fibroblasts, and its in vivo effects in a well‐established rat model of patellar tendon donor site injury. Results: It was found that gene expression of COL3A1 and decorin in cultured tendon fibroblasts was significantly increased by RD, but that COL1A1 was not affected. In vivo studies showed that RD increased blood vessels in the wound but did not significantly affect the expression of COL1A1, COL3A1 and decorin at day 14 post‐injury. The ultimate tensile stress of the healing tendon was not significantly improved by either local injection or oral administration of hot water extract of RD (P > 0.05). Conclusion: The present findings imply that RD per se does not significantly improve tendon healing. Further investigation of RD in a herbal formula may be necessary to test its efficacy in tendon injuries.     

Radial shock waves effectively introduced NF‐kappa B decoy into rat achilles tendon cells in vitro

The purpose of this study was to test if radial shock waves could enhance the introduction of nuclear factor‐kappa B (NF‐κB) decoy oligodeoxynucleotides, which is reported to markedly inhibit NF‐κB activation and suppress pro‐inflammatory cytokine gene expression, using rat Achilles tendon cells. In the presence of NF‐κB decoy labeled with or without fluorescein isothiocyanate (FITC) in culture media, radial shock waves were applied to the tendon cells in variable conditions and cultivated for 24 h. The transfection rate was assessed by counting FITC‐positive cells, and IL‐1–induced NF‐κB activation in the cells was assessed. Radial shock waves significantly enhanced introduction of NF‐κB decoy‐FITC into the tendon cells. IL‐1–induced NF‐κB activation was significantly inhibited by pretreatment of the cells with NF‐κB decoy combined with radial shock wave exposure. The present study demonstrated the effectiveness of radial shock waves on introduction of NF‐κB decoy into tendon cells. Radial shock wave treatment combined with local NF‐κB decoy administration could be a novel therapeutic strategy for chronic tendinopathy. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1078–1083, 2010     

Expression of transforming growth factor β isoforms and their roles in tendon healing

Transforming growth factor β (TGF‐β) plays active roles in tendon healing. However, the differential effects of TGF‐β isoforms on tendon healing have not been investigated. In cultured tendon fibroblasts, we tested the effects of TGF‐β1, β2, and β3 on the mRNA levels of COL1A1 and COL3A1 by quantitative real‐time polymerase chain reaction. We also investigated the expression of TGF‐β isoforms, TGF‐β receptors, procollagen Type I and Type III in a rat model of tendon healing. We found that TGF‐β3 exhibited the highest potency in stimulating COL1A1 and COL3A1. TGF‐β1 exerted antagonistic effects to TGF‐β2 and β3. All TGF‐β isoforms and procollagen Type I were confined to the edges of the healing tendon at day 28 postinjury. Our results indicated that interaction of TGF‐β isoforms exist in the regulation of collagen synthesis in tendon fibroblasts. Their effects may be further complicated by uneven spatial distribution of TGF‐β and TGF‐β receptors in healing tendons.     

Temporal growth factor release from platelet‐rich plasma,trehalose lyophilized platelets,and bone marrow aspirate and their effect on tendon and ligament gene expression

Platelet‐rich plasma (PRP) has generated substantial interest for tendon and ligament regeneration because of the high concentrations of growth factors in platelet α‐granules. This study compared the temporal release of growth factors from bone marrow aspirate (BMA), PRP, and lyophilized platelet product (PP), and measured their effects on tendon and ligament gene expression. Blood and BMA were collected and processed to yield PRP and plasma. Flexor digitorum superficialis tendon (FDS) and suspensory ligament (SL) explants were cultured in 10% plasma in DMEM (control), BMA, PRP, or PP. TGF‐β1 and PDGF‐BB concentrations were determined at 0, 24, and 96 h of culture using ELISA. Quantitative RT‐PCR for collagen types I and III (COL1A1, COL3A1), cartilage oligomeric matrix protein (COMP), decorin, and matrix metalloproteinases‐3 and 13 (MMP‐3, MMP‐13) was performed. TGF‐β1 and PDGF‐BB concentrations were highest in PRP and PP. Growth factor quantity was unchanged in BMA, increased in PRP, and decreased in PP over 4 days. TGF‐β1 and platelet concentrations were positively correlated. Lyophilized PP and PRP resulted in increased COL1A1:COL3A1 ratio, increased COMP, and decreased MMP‐13 expression. BMA resulted in decreased COMP and increased MMP‐3 and MMP‐13 gene expression. Platelet concentration was positively correlated with COL1A1, ratio of COL1A1:COL3A1, and COMP, and negatively correlated with COL3A1, MMP‐13, and MMP‐3. White blood cell concentration was positively correlated with COL3A1, MMP3, and MMP13, and negatively correlated with a ratio of COL1A1:COL3A1, COMP, and decorin. These findings support further in vivo investigation of PRP and PP for treatment of tendonitis and desmitis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1033–1042, 2009     

Do antioxidants inhibit oxidative‐stress‐induced autophagy of tenofibroblasts?

Recent research on tendinopathy has focused on its relationship to programmed cell death. Increased autophagy has been observed in ruptured rotator cuff tendon tissues, suggesting a causal relationship. We investigated whether autophagy occurs in human rotator cuff tenofibroblast death induced by oxidative stress and whether antioxidants protect against autophagic cell death. We used H₂O₂ (0.75 mM) as oxidative stressor, cyanidin (100 µg/ml) as antioxidant, zVAD (20 µM) as apoptosis inhibitor, and 3‐MA (10 mM) as autophagy inhibitor. We evaluated cell viability and known autophagic markers: LC3‐II expression, GFP‐LC3 puncta formation, autolysosomes, and Atg5‐12 and Beclin 1 expression. H₂O₂ exposure increased the rates of cell death, LC3‐II expression, GFP‐LC3 puncta formation, and autolysosomes. After we induced apoptosis arrest using zVAD, H₂O₂ exposure still induced cell death, LC3‐II expression, and GFP‐LC3 puncta formation. H₂O₂ exposure also increased Atg5‐12 and Beclin 1 expressions, indicating autophagic cell death. However, cyanidin treatment reduced H₂O₂‐induced cell death, LC3‐II expression, GFP‐LC3 puncta formation, and autolysosomes. Cyanidin and 3‐MA similarly reduced the cell‐death rate, and Atg5‐12 and Beclin 1 expression. This study demonstrated that H₂O₂, an oxidative stressor, induces autophagic cell death in rotator cuff tenofibroblasts, and that cyanidin, a natural antioxidant, inhibits autophagic cell death. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:937–943, 2014.     

Dose–response effect of an intra‐tendon application of recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB) in a rat Achilles tendinopathy model

The purpose of this study was to assess whether intra‐tendon delivery of recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB) would improve Achilles tendon repair in a rat collagenase‐induced tendinopathy model. Seven days following collagenase induction of tendinopathy, one of four intra‐tendinous treatments was administered: (i) Vehicle control (sodium acetate buffer), (ii) 1.02 µg rhPDGF‐BB, (iii) 10.2 µg rhPDGF‐BB, or (iv) 102 µg rhPDGF‐BB. Treated tendons were assessed for histopathological (e.g., proliferation, tendon thickness, collagen fiber density/orientation) and biomechanical (e.g., maximum load‐to‐failure and stiffness) outcomes. By 7 days post‐treatment, there was a significant increase in cell proliferation with the 10.2 and 102 µg rhPDGF‐BB‐treated groups (p = 0.049 and 0.015, respectively) and in thickness at the tendon midsubstance in the 10.2 µg of rhPDGF‐BB group (p = 0.005), compared to controls. All groups had equivalent outcomes by Day 21. There was a dose‐dependent effect on the maximum load‐to‐failure, with no significant difference in the 1.02 and 102 µg rhPDGF‐BB doses but the 10.2 µg rhPDGF‐BB group had a significant increase in load‐to‐failure at 7 (p = 0.003) and 21 days (p = 0.019) compared to controls. The rhPDGF‐BB treatment resulted in a dose‐dependent, transient increase in cell proliferation and sustained improvement in biomechanical properties in a rat Achilles tendinopathy model, demonstrating the potential of rhPDGF‐BB treatment in a tendinopathy application. Consequently, in this model, data suggest that rhPDGF‐BB treatment is an effective therapy and thus, may be an option for clinical applications to treat tendinopathy. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 413–420, 2013     

Expression of interleukin‐1β, cyclooxygenase‐2, and prostaglandin E2 in a rotator cuff tear in rabbits

We investigated the specific factors related to shoulder pain due to a rotator cuff tear using a model in rabbits. A rotator cuff tear was surgically created, and the expression of interleukin‐1β (IL‐1β), prostaglandin E2 (PGE2), and cyclooxygenase‐2 (COX‐2) was analyzed. In the supernatant of the tissue culture of the torn tendon, IL‐1β production was detected. The amount of IL‐1β was highest 1 day after injury, and then decreased gradually to 21 days. PGE2, the mediator of pain and the product of COX‐2, was also detected in the supernatant of the tissue culture. The production of PGE2 significantly increased to 7 days after injury, and then decreased to 21 days. RT‐PCR analysis confirmed the mRNA expression of IL‐1β and COX‐2 in the torn tendon. Immunohistochemical study demonstrated that cells in the tendon stump were immunopositive for IL‐1β and COX‐2. Furthermore, in the affected joint, articular chondrocytes in the remote area from the tear expressed COX‐2 strongly. When the rotator cuff is torn, IL‐1β is produced in the torn tendon, and stimulates the expression of COX‐2 in not only the torn tendon but also in articular chondrocytes. The COX‐2 then produces PGE2, which would mediate shoulder pain. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:92–97, 2007     

Impact of direct cell co‐cultures on human adipose‐derived stromal cells and nucleus pulposus cells

Biologic and cellular treatment strategies aiming for curing intervertebral disc degeneration (IDD) have been proposed recently. Given the convenient availability and expansion potential, adipose‐derived stromal cells (ADSCs) might be an ideal cell candidate. However, the interaction between ADSCs and nucleus pulposus (NP) cells still remains ambiguous, especially in direct co‐cultures of the two types of cells. Nevertheless, NP markers in ADSCs after co‐cultures were unidentified. Here, we addressed the interaction of human ADSCs and NP cells in a direct co‐culture system for the first time. As a result, ADSCs could differentiate to the NP cell phenotype with a significant up‐regulated expression of multiple genes and proteins in extracellular matrix (ECM) (SOX9, COL2A1, ACAN, and COL6A2), relative NP markers (FOXF1, PAX1, CA12, and HBB) and pertinent growth factors (CDMP‐1, TGF‐β1, IGF‐1, and CTGF). Moreover, the gene expression of COL2A1, ACAN, and COL6A2 of degenerate NP cells was also up‐regulated. Collectively, these results suggest that direct co‐cultures of ADSCs and NP cells may exert a reciprocal impact, that is, both stimulating ADSCs differentiation to the NP cell phenotype and inducing NP cells to regain functional phenotype. Accordingly, ADSCs might be a potential candidate in the development of cellular treatment strategies for IDD. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1804–1813, 2013     

Platelet‐rich plasma protects rotator cuff‐derived cells from the deleterious effects of triamcinolone acetonide

Triamcinolone acetonide (TA) injections are widely used to treat enthesopathy, but they may induce adverse effects such as tendon impairment and rupture. Platelet‐rich plasma (PRP) is a blood fraction containing high platelet concentrations and various growth factors that play a role in tissue repair processes. The purpose of this study is to investigate whether TA has deleterious effects on human rotator cuff‐derived cells, and if PRP can protect these cells from the effects of TA. Human rotator cuff‐derived cells were cultured with and without TA and PRP, and the culture without any additive served as the control. Cell morphology was assessed at days 7 and 21. Cell viability was evaluated at days 1, 7, 14, and 21 by a water‐soluble tetrazolium salt assay. Induction of apoptosis was measured by immunofluorescence staining and flow cytometry at day 7. Induction of cleaved caspase‐3 was measured by immunofluorescence staining at day 7. The cells cultured with TA had a flattened and polygonal shape at day 7. The cells cultured with both TA and PRP were similar in appearance to control cells. Exposure to TA also significantly decreased cell viability, but cell viability did not decrease when PRP was added along with TA. The number of apoptotic cells increased with TA exposure, while addition of PRP prevented cell apoptosis. In conclusion, the deleterious effect of TA was prevented by PRP, which can be used as a protective agent for patients receiving local TA injections. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 976–982, 2013     

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     

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     

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.     

Mechanical loading modulates glutamate receptor subunit expression in bone

The cellular mechanisms coupling mechanical loading with bone remodeling remain unclear. In the CNS, the excitatory amino acid glutamate (Glu) serves as a potent neurotransmitter exerting its effects via various membrane Glu receptors (GluR). Nerves containing Glu exist close to bone cells expressing functional GluRs. Demonstration of a mechanically sensitive glutamate/aspartate transporter protein and the ability of glutamate to stimulate bone resorption in vitro suggest a role for glutamate linking mechanical load and bone remodeling. We used immunohistochemical techniques to identify the expression of N-methyl-d-aspartate acid (NMDA) and non-NMDA (AMPA or kainate) ionotropic GluR subunits on bone cells in vivo. In bone sections from young adult rats, osteoclasts expressed numerous GluR subunits including AMPA (GluR2/3 and GluR4), kainic acid (GluR567) and NMDA (NMDAR2A, NMDAR2B and NMDAR2C) receptor subtypes. Bone lining cells demonstrated immunoexpression for NMDAR2A, NMDAR2B, NMDAR2C, GluR567, GluR23, GluR2 and GluR4 subunits. Immunoexpression was not evident on osteocytes, chondrocytes or vascular channels. To investigate the effects of mechanical loading on GluR expression, we used a Materials Testing System (MTS) to apply 10 N sinusoidal axial compressive loads percutaneously to the right limbs (radius/ulna, tibia/fibula) of rats. Each limb underwent 300-load cycles/day (cycle rate, 1 Hz) for 4 consecutive days. Contralateral, non-loaded limbs served as controls. Mechanically loaded limbs revealed a load-induced loss of immunoexpression for GluR2/3, GluR4, GluR567 and NMDAR2A on osteoclasts and NMDAR2A, NMDAR2B, GluR2/3 and GluR4 on bone lining cells. Both neonatal rabbit and rat osteoclasts were cultured on bone slices to investigate the effect of the NMDA receptor antagonist, MK801, and the AMPA/kainic acid receptor antagonist, NBQX, on osteoclast resorptive activity in vitro. The inhibition of resorptive function seen suggested that both NMDAR and kainic acid receptor function are required for normal osteoclast function. While the exact role of ionotropic GluRs in skeletal tissue remains unclear, the modulation of GluR subunit expression by mechanical loading lends further support for participation of Glu as a mechanical loading effector. These ionotropic receptors appear to be functionally relevant to normal osteoclast resorptive activity.     

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