Adult human articular cartilage harbors a population of CD166+ mesenchymal stem cell-like progenitors that become more numerous during osteoarthritis (OA). While their role is not well understood, here we report that they are indeed part of cellular clusters formed in OA cartilage, which is a pathological hallmark of this disease. We hypothesize that these cells, termed OA mesenchymal stem cells (OA-MSCs), contribute to OA pathogenesis. To test this hypothesis, we generated and characterized multiple clonally derived stable/immortalized human OA-MSC cell lines, which exhibited the following properties. Firstly, two mesenchymal stem cell populations exist in human OA cartilage. While both populations are multi-potent, one preferentially undergoes chondrogenesis while the other exhibits higher osteogenesis potential. Secondly, both OA-MSCs exhibit significantly higher expression of hypertrophic OA cartilage markers COL10A1 and RUNX2, compared to OA chondrocytes. Induction of chondrogenesis in OA-MSCs further stimulated COL10A1 expression and MMP-13 release, suggesting that they contribute to OA phenotypes. Finally, knocking down RUNX2 is insufficient to inhibit COL10A1 in OA-MSCs and also requires simultaneous knockdown of NOTCH1 thereby suggesting altered gene regulation in OA stem cells in comparison to chondrocytes. Overall, our findings suggest that OA-MSCs may drive pathogenesis of cartilage degeneration and should therefore be a novel cell target for OA therapy.

SHP2 is a ubiquitously expressed protein tyrosine phosphatase, which is involved in many signaling pathways to regulate the skeletal development. In endochondral ossification, SHP2 is known to modify the osteogenic fate of osteochondroprogenitors and to impair the osteoblastic transdifferentiation of hypertrophic chondrocytes. However, how SHP2 regulates osteoblast differentiation in intramembranous ossification remains incompletely understood. To address this question, we generated a mouse model to ablate SHP2 in the Prrx1-expressing mesenchymal progenitors by using “Cre-loxP”-mediated gene excision and examined the development of calvarial bone, in which the main process of bone formation is intramembranous ossification. Phenotypic characterization showed that SHP2 mutants have severe defects in calvarial bone formation. Cell lineage tracing and in situ hybridization data showed less osteoblast differentiation of mesenchymal cells and reduced osteogenic genes expression, respectively. Further mechanistic studies revealed enhanced TGFβ and suppressed BMP2 signaling in SHP2 ablated mesenchymal progenitors and their derivatives. Our study uncovered the critical role of SHP2 in osteoblast differentiation through intramembranous ossification and might provide a potential target to treat craniofacial skeleton disorders.

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor (OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2 (encoded by Ptpn11 ) affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using “Cre-loxP”-mediated gene excision. SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1 , and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, qRT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.

Meniscus injuries are among the most common orthopedic injuries. Tears in the inner one-third of the meniscus heal poorly and present a significant clinical challenge. In this study, we hypothesized that progenitor cells from healthy human articular cartilage (chondroprogenitor cells [C-PCs]) may be more suitable than bone-marrow mesenchymal stem cells (BM-MSCs) to mediate bridging and reintegration of fibrocartilage tissue tears in meniscus. C-PCs were isolated from healthy human articular cartilage based on their expression of mesenchymal stem/progenitor marker activated leukocyte cell adhesion molecule (ALCAM) (CD166). Our findings revealed that healthy human C-PCs are CD166+, CD90+, CD54+, CD106- cells with multilineage differentiation potential, and elevated basal expression of chondrogenesis marker SOX-9. We show that, similar to BM-MSCs, C-PCs are responsive to the chemokine stromal cell-derived factor-1 (SDF-1) and they can successfully migrate to the area of meniscal tissue damage promoting collagen bridging across inner meniscal tears. In contrast to BM-MSCs, C-PCs maintained reduced expression of cellular hypertrophy marker collagen X in monolayer culture and in an explant organ culture model of meniscus repair. Treatment of C-PCs with SDF-1/CXCR4 pathway inhibitor AMD3100 disrupted cell localization to area of injury and prevented meniscus tissue bridging thereby indicating that the SDF-1/CXCR4 axis is an important mediator of this repair process. This study suggests that C-PCs from healthy human cartilage may potentially be a useful tool for fibrocartilage tissue repair/regeneration because they resist cellular hypertrophy and mobilize in response to chemokine signaling. Stem Cells 2019;37:102-114.

Transdifferentiation of hypertrophic chondrocytes into bone-forming osteoblasts has been reported, yet the underlying molecular mechanism remains incompletely understood. SHP2 is an ubiquitously expressed cytoplasmic protein tyrosine phosphatase. SHP2 loss-of-function mutations in chondroid cells are linked to metachondromatosis in humans and mice, suggesting a crucial role for SHP2 in the skeleton. However, the specific role of SHP2 in skeletal cells has not been elucidated. To approach this question, we ablated SHP2 in collagen 2α1(Col2α1)-Cre- and collagen 10α1(Col10α1)-Cre-expressing cells, predominantly proliferating and hypertrophic chondrocytes, using "Cre-loxP"-mediated gene excision. Mice lacking SHP2 in Col2α1-Cre-expressing cells die at mid-gestation. Postnatal SHP2 ablation in the same cell population caused dwarfism, chondrodysplasia and exostoses. In contrast, mice in which SHP2 was ablated in the Col10α1-Cre-expressing cells appeared normal but were osteopenic. Further mechanistic studies revealed that SHP2 exerted its influence partly by regulating the abundance of SOX9 in chondrocytes. Elevated and sustained SOX9 in SHP2-deficient hypertrophic chondrocytes impaired their differentiation to osteoblasts and impaired endochondral ossification. Our study uncovered an important role of SHP2 in bone development and cartilage homeostasis by influencing the osteogenic differentiation of hypertrophic chondrocytes and provided insight into the pathogenesis and potential treatment of skeletal diseases, such as osteopenia and osteoporosis.

Genes that regulate osteoclast (OC) development and function in both physiologic and disease conditions remain incompletely understood. Shp2 (the Src homology‐2 domain containing protein tyrosine phosphatase 2), a ubiquitously expressed cytoplasmic protein tyrosine phosphatase, is implicated in regulating M‐CSF and receptor activator of nuclear factor‐κB ligand (RANKL)‐ evoked signaling; its role in osteoclastogenesis and bone homeostasis, however, remains unknown. Using a tissue‐specific gene knockout approach, we inactivated Shp2 expression in murine OCs. Shp2 mutant mice are phenotypically osteopetrotic, featuring a marked increase of bone volume (BV)/total volume (TV) (+42.8%), trabeculae number (Tb.N) (+84.1%), structure model index (+119%), and a decrease of trabecular thickness (Tb.Th) (‐34.1%) and trabecular spacing (Tb.Sp) (‐41.0%). Biochemical analyses demonstrate that Shp2 is required for RANKL‐induced formation of giant multinucleated OCs by up‐regulating the expression of nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1), a master transcription factor that is indispensable for terminal OC differentiation. Shp2 deletion, however, has minimal effect on M‐CSF‐dependent survival and proliferation of OC precursors. Instead, its deficiency aborts the fusion of OC precursors and formation of multinucleated OCs and decreases bone matrix resorption. Moreover, pharmacological intervention of Shp2 is sufficient to prevent preosteoclast fusion in vitro. These findings uncover a novel mechanism through which Shp2 regulates osteoclastogenesis by promoting preosteoclast fusion. Shp2 or its signaling partners could potentially serve as pharmacological targets to regulate the population of OCs locally and/or systematically, and thus treat OC‐related diseases, such as periprosthetic osteolysis and osteoporosis.—Zhou, Y., Mohan, A., Moore, D. C., Lin, L., Zhou, F. L., Cao, J., Wu, Q., Qin, Y.‐X., Reginato, A. M., Ehrlich, M. G., Yang, W. SHP2 regulates osteoclastogenesis by promoting preosteoclast fusion. FASEB J. 29, 1635‐1645 (2015). www.fasebj.org

Nature 499, 491–495 (2013); doi:10.1038/nature12396 After publication of this Letter, we became aware that we had not reported the details of construction and validation of our floxed conditional deletion Ptpn11 allele. These details are presented in the Supplementary Methods and Supplementary Fig. 1 of this Corrigendum.

Objective—To determine if supplemental intra-articular alpha-2 macroglobulin (A2M) has a chondroprotective effect in a rat OA model. Methods—A2M was identified as a potential therapeutic agent by comparing A2M concentrations in serum, synovial fluid (SF), and cartilage from normal and osteoarthritic (OA) patients by Western blotting, mass spectrometry, ELISA, and immunohistochemistry (IHC). The effects of A2M on IL-1-induced cartilage catabolic enzymes were evaluated by Luminex and ELISA in cultured chondrocytes. In vivo effects on cartilage degeneration and MMP-13 concentration were evaluated in male rats (N=120) randomized to four treatments: (1) CLT +saline, (2) ACLT+A2M (1IU/kg), (3) ACLT+A2M (2IU/kg) or (4) sham surgery+saline. Intraarticular injections were given for 6 weeks. The concentration of MMP-13 in SF lavages was Correspondence to: Lei Wei, 401-793-8384(O), (401)444-5872(Fax) Lei_Wei@brown.edu. †These authors contributed in equal measure as the first author. Authors’ contributions: Shaowei Wang participated in the study design, wrote the manuscript, performed most of the experiments and analyzed data. Jingming Zhou, Jing Zhang, and Kai Li performed some of the experiments and analyzed data. Mary Goldring provided the C28 cell line and provided advice on its use. Richard Terek, Braden C. Fleming, Xiaochun Wei and Michael G. Ehrlich provided the human samples. Xiaochun Wei, Jingming Zhou, Jing Zhang, Kai Li, Qian Chen, Richard Terek, Braden C. Fleming, Mary Goldring, Michael G. Ehrlich, and Ge Zhang participated in the interpretation of the data and/or revised the manuscript critically. Lei Wei conceived of the study, participated in its design and data analysis, and revised the manuscript carefully and critically. All authors have read and approved the final manuscript. Disclosures: We have nothing to disclose. NIH Public Access Author Manuscript Arthritis Rheumatol. Author manuscript; available in PMC 2015 July 01. Published in final edited form as: Arthritis Rheumatol. 2014 July ; 66(7): 1843–1853. doi:10.1002/art.38576. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript measured using ELISA. OA-related gene expression was quantified by RT-qPCR. Histology was performed to grade OA. Results—In both normal and OA patients, the levels of A2M were lower in SF compared to serum, and MMP-13 was higher in SF than serum of OA patients. In vitro, A2M inhibited the induction of MMP-13 by IL-1 in a dose-dependent manner in human chondrocytes. In the rat ACLT OA model, supplemental intra-articular injection of A2M reduced the concentration of MMP-13 in SF, had a favorable effect on OA-related gene expression, and attenuated OA progression. Conclusion—A2M is a plasma protease inhibitor that is not present in sufficient concentrations to inactivate the high concentrations of catabolic factors found in OA SF. Our findings suggest that supplemental intra-articular A2M provides chondral protection for post traumatic OA.

ObjectiveTo determine if supplemental intraarticular α2‐macroglobulin (α2M) has a chondroprotective effect in a rat model of osteoarthritis (OA).MethodsUsing Western blotting, mass spectrometry, enzyme‐linked immunosorbent assay (ELISA), and immunohistochemistry, α2M was identified as a potential therapeutic agent through a comparison of α2M concentrations in serum, synovial fluid (SF), and cartilage from normal subjects and patients with OA. In cultured chondrocytes, the effects of α2M on interleukin‐1 (IL‐1)–induced cartilage catabolic enzymes were evaluated by Luminex assay and ELISA. In vivo effects on cartilage degeneration and matrix metalloproteinase 13 (MMP‐13) concentration were evaluated in male rats (n = 120) randomized to 1 of 4 treatments: 1) anterior cruciate ligament transection (ACLT) and saline injections, 2) ACLT and 1 IU/kg injections of α2M, 3) ACLT and 2 IU/kg injections of α2M, or 4) sham operation and saline injections. Rats were administered intraarticular injections for 6 weeks. The concentration of MMP‐13 in SF lavage fluid was measured using ELISA. OA‐related gene expression was quantified by real‐time quantitative polymerase chain reaction. The extent of OA progression was graded by histologic examination.ResultsIn both normal subjects and OA patients, α2M levels were lower in SF as compared to serum, and in OA patients, MMP‐13 levels were higher in SF than in serum. In vitro, α2M inhibited the induction of MMP‐13 by IL‐1 in a dose‐dependent manner in human chondrocytes. In the rat model of ACLT OA, supplemental intraarticular injection of α2M reduced the concentration of MMP‐13 in SF, had a favorable effect on OA‐related gene expression, and attenuated OA progression.ConclusionThe plasma protease inhibitor α2M is not present in sufficient concentrations to inactivate the high concentrations of catabolic factors found in OA SF. Our findings suggest that supplemental intraarticular α2M provides chondral protection in posttraumatic OA.

BACKGROUND:Work-hour restrictions and increased supervision requirements have altered the clinical experience of orthopaedic surgery residents, while the specialty's body of knowledge and requisite skill set continue to expand. This dilemma means that the duration and practice experience of the traditional orthopaedic residency may not meet the needs of today's trainees. For the past eighteen years, however, residency training in the Department of Orthopaedic Surgery at Brown University has included a mandatory postgraduate year six (PGY6) trauma fellowship-modeled year, during which trainees are conferred full staff admitting and operating privileges, with time allotted for completing research. They are supervised by senior attending staff, with increasing autonomy as the year progresses. A formal, critical analysis of this transition-to-practice training model in orthopaedics has not previously been described.METHODS:An anonymous thirty-one-item questionnaire was distributed to all practicing graduates of the six-year Brown University Orthopaedic Surgery training program (n = 69). A 5-point Likert scale was used to assess attitudinal questions. An independent-sample t test was used to compare the responses of pre-duty-hour trainees with those of post-duty-hour trainees, with a p value of <0.05 utilized for significance.RESULTS:All sixty-nine practicing graduates of the Brown University PGY6 trauma fellowship completed the survey (100% response rate). Most graduates (78.2%) would choose to complete the PGY6 year if they had to do residency again, and 72.4% would recommend trauma fellowship-modeled training to residents beginning their training. Trainees who completed residency during or after the imposed 2003 Accreditation Council for Graduate Medical Education duty-hour restrictions (79.3%) were significantly more likely (p = 0.014) to rank the PGY6 year as their most valuable training year compared with trainees who completed residency prior to duty-hour restrictions (50.0%). Nearly half of the graduates (46.4%) thought that the PGY6 fellowship year was financially burdensome.CONCLUSIONS:The unique trauma fellowship-modeled sixth year of orthopaedic surgery training at Brown University was thought to be a valuable training experience by a large majority of graduates, although nearly half thought that the year was financially burdensome. These data suggest that a trauma fellowship-based sixth year of independent yet structured training has the potential to enhance orthopaedic education and could become an alternative standard given the current requirements imposed upon surgical residency training. These results may help guide further discussion among orthopaedic training programs to determine the optimal model for orthopaedic residency education in the twenty-first century.

If left uncorrected, valgus ankle deformity in multiple hereditary exostoses can cause significant disability in skeletally immature children and in adults. Various management methods have been described, including hemiepiphyseal stapling, transphyseal screw placement, fibular-Achilles tenodesis, distal tibial osteotomy, and ablative epiphyseodesis. In this article, we report the cases of 3 skeletally immature children who had undergone hemiepiphyseal stapling of the medial distal tibial epiphysis for correction of valgus ankle deformity in multiple hereditary exostoses. Correction of the tibiotalar axis, in relation to chronological and bone age, was evaluated. Hemiepiphyseal stapling of the medial distal tibial epiphysis provides ipsilateral corrective potential while allowing staple removal for reversal of growth retardation. This procedure is useful in the management of ankle valgus in multiple hereditary exostoses.

ObjectiveOsteoarthritis (OA) is associated with obesity, although this relationship remains unclear. Proposed etiologies of OA in obesity include mechanical loading of malaligned joints and possible toxicity of dietary fat. The hypothesis tested in the present study was that increased dietary fat worsens OA in both malaligned and normal joints, detected by biochemical and histological cartilage markers.Method83 New Zealand white rabbits were divided among two conditions related to OA: bowing of the knee and a 14% kcal vs 47.8% kcal fat diet. Rabbit weights and knee angles were compared throughout the experiment. At 28 and 38 weeks, intra-articular forces were measured, animals sacrificed, and knee cartilage examined for histological changes, glycosaminoglycan content, 35S uptake, and aggrecanase-1 expression.ResultsThere were no differences in animal weights or intra-articular forces between the two diets. Despite increased fat content in their diet, animals on the 47.8% kcal fat diet did not gain excess weight. Representative histology showed atypical shearing of articular cartilage among animals on the high fat diet. Animals on the 47.8% kcal fat diet had suppression of protein synthesis compared to the 14% kcal fat diet: lower glycosaminoglycan content and aggrecanase-1 expression in all knee compartments at both times, and lower 35S uptake at 38 weeks.ConclusionThese results suggest dietary fat, independent of animal weight, results in altered chondrocyte function. Increased dietary fat was associated with changes in rabbit cartilage in vivo and appears to be a risk factor for the development of OA.

Cellular interactions at the interface between bone and a prosthetic device plays an important role in total joint reconstruction success or failure. Aseptic loosening is a typical long-term complication encountered due to a failure at this interface. A number of factors have been shown to influence cell mitogenic and morphological behavior including load and stability [1] surface topography and surface chemistry [2]. The selection of suitable surface coatings on a prosthetic device would be facilitated if the nature of the interaction of bone cells to surface could be predicted from appropriate behavior of cells in tissue culture [3]. Molecular biology techniques now enable investigators to study cell-substrate interactions and gene expression at the mRNA level.

Prewarming operating rooms has been shown to limit hypothermia in pediatric surgical patients but may be associated with extreme discomfort for surgeons. We examined the effect of prewarming operating rooms on core temperatures during knee and hip arthroplasties. Sixty-six patients were randomized to the prewarmed group at 24°C or control group at 17°C. The prewarmed group core temperature (mean, 36.14°C) before active warming was significantly higher (P = .018) than that of the control group (mean, 35.83°C). By the start of surgery, the difference was 36.01°C prewarmed vs 35.83°C control, P = .038. There was no significant difference in the last recorded mean temperatures between groups: 36.35°C (prewarmed) vs 36.16°C (control). A prewarmed operating room for adults undergoing knee or hip arthroplasty had minimal effect on preventing intraoperative hypothermia.

Objective: The objective of this study was to analyze the appropriateness of transfer of patients with orthopaedic injuries to a Level I trauma center from surrounding Level II to IV centers.Design: A prospective study was conducted over a 5-month period by collecting data on all orthopaedic patients being transferred to our facility. All transfer diagnoses were designated as appropriate or inappropriate. Patient demographics were calculated.Setting: The transfer of patients occurred from 23 Level II to IV hospitals to a Level I trauma center. These hospitals service 1 to 1.5 million people a year.Patients/Participants Main Outcome Measurements: All patients transferred with orthopaedic injuries were recorded. Patient variables such as transfer diagnosis, age, gender, insurance status, time of arrival, day of transfer, transferring and accepting physicians, previous imaging studies, and patient disposition were recorded. Outcome measurements included chi(2) tests to determine variation in demographics based on insurance and appropriateness of transfer. Multivariate regression analysis was also performed to determine influence of individual patient variables on the main outcome variable: appropriateness of transfer.Results: Two hundred sixteen patients were transferred of which we considered 52% inappropriate. Sixty-eight percent of transfers occurred between 6: 00 PM and 5: 59 AM and 60% of all transfers were over the weekend. Also, 69% of inappropriate transfers were discharged directly from the emergency department. Insurance was an independent factor affecting appropriateness of transfer. A larger percentage of inappropriate patients transferred were uninsured. The inappropriate patient who was transferred had more likelihood of being uninsured than insured. Moreover, there was a significantly higher percentage of inappropriate uninsured patients transferred after hours and over the weekend as compared with insured patients. More than 97% of inappropriate transfers were accepted by the emergency department physician without communication with the on-call orthopaedist at our facility.Conclusions: There is a trend among community hospitals to transfer uninsured patients with benign orthopaedic injuries in-appropriately to a Level I trauma center. This effect is magnified on weekends and at night. Strict regulation of the Emergency Labor Act and better communication between Level II to IV hospitals and Level I orthopaedic surgeons can decrease the inappropriate transfer of patients and reduce the burden on our healthcare system.