Human Cartilage-Derived Progenitors Resist Terminal Differentiation and Require CXCR4 Activation to Successfully Bridge Meniscus Tissue Tears.

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.

Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling.

The tyrosine phosphatase SHP2, encoded by PTPN11, is required for the survival, proliferation and differentiation of various cell types. Germline activating mutations in PTPN11 cause Noonan syndrome, whereas somatic PTPN11 mutations cause childhood myeloproliferative disease and contribute to some solid tumours. Recently, heterozygous inactivating mutations in PTPN11 were found in metachondromatosis, a rare inherited disorder featuring multiple exostoses, enchondromas, joint destruction and bony deformities. The detailed pathogenesis of this disorder has remained unclear. Here we use a conditional knockout (floxed) Ptpn11 allele (Ptpn11(fl)) and Cre recombinase transgenic mice to delete Ptpn11 specifically in monocytes, macrophages and osteoclasts (lysozyme M-Cre; LysMCre) or in cathepsin K (Ctsk)-expressing cells, previously thought to be osteoclasts. LysMCre;Ptpn11(fl/fl) mice had mild osteopetrosis. Notably, however, CtskCre;Ptpn11(fl/fl) mice developed features very similar to metachondromatosis. Lineage tracing revealed a novel population of CtskCre-expressing cells in the perichondrial groove of Ranvier that display markers and functional properties consistent with mesenchymal progenitors. Chondroid neoplasms arise from these cells and show decreased extracellular signal-regulated kinase (ERK) pathway activation, increased Indian hedgehog (Ihh) and parathyroid hormone-related protein (Pthrp, also known as Pthlh) expression and excessive proliferation. Shp2-deficient chondroprogenitors had decreased fibroblast growth factor-evoked ERK activation and enhanced Ihh and Pthrp expression, whereas fibroblast growth factor receptor (FGFR) or mitogen-activated protein kinase kinase (MEK) inhibitor treatment of chondroid cells increased Ihh and Pthrp expression. Importantly, smoothened inhibitor treatment ameliorated metachondromatosis features in CtskCre;Ptpn11(fl/fl) mice. Thus, in contrast to its pro-oncogenic role in haematopoietic and epithelial cells, Ptpn11 is a tumour suppressor in cartilage, acting through a FGFR/MEK/ERK-dependent pathway in a novel progenitor cell population to prevent excessive Ihh production.

SHP2 regulates osteoclastogenesis by promoting preosteoclast fusion.

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.

Prewarming operating rooms for prevention of intraoperative hypothermia during total knee and hip arthroplasties.

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.

SHP2 regulates intramembranous ossification by modifying the TGFß and BMP2 signaling pathway.

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 TGFß 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.

Subtalar release in clubfeet: a retrospective study of 10-year outcomes.

BACKGROUND: Optimal management of congenital talipes equinovarus continues to be controversial. There is a dramatically renewed emphasis on non-operative management partly because there has been a high recurrence rate among operated feet. Our hypothesis is that early, extensive subtalar ligament release as the cornerstone of aggressive hindfoot realignment prevents recurrence and retains mobility. MATERIALS AND METHODS: Twenty-two congenital clubfeet (14 patients) corrected by one surgeon were evaluated using two validated patient-based outcome instruments, dynamic pedobarographic analysis, hindfoot mobility, and weightbearing radiographs. Pedobarographic analysis consisted of quantifying peak plantar forces and pressures during the gait cycle in 22 corrected feet and 24 control feet using the FSCAN in-shoe device. RESULTS: The mean age at surgery was 8 months and mean followup was 10 years. No patients experienced recurrence of deformity. Reported foot function and satisfaction were very high for all patients and were comparable to reported normal population values. AP and lateral talocalcaneal angles for each foot were within normal limits for age. Hindfoot range of motion, including dorsiflexion, was preserved in all feet. Peak regional forces throughout the gait cycle and plantar pressures at foot flat were mildly, but statistically significantly, higher in the midfoot of corrected feet suggesting slight flattening of the arch. One patient had tendon transfers for bilateral calcaneal deformity and one patient had surgical correction of a bilateral valgus deformity. CONCLUSION: Aggressive hindfoot realignment provides definitive treatment of an equinovarus deformity, but care must be taken to avoid overcorrection.

Molecular characterization of mesenchymal stem cells in human osteoarthritis cartilage reveals contribution to the OA phenotype.

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 regulates skeletal cell fate by modifying SOX9 expression and transcriptional activity.

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.

Epiphysiodesis with infusion of stromal cell-derived factor-1 in rabbit growth plates.

BACKGROUND: The mechanism of physeal closure is poorly understood, although both mechanical and biological factors may play a role in the process. In this study, we evaluated the effect of the application of a chemokine stromal cell-derived factor-1 (SDF-1) to rabbit physes in vivo with regard to growth inhibition. METHODS: A continuous infusion system consisting of a fenestrated catheter and an osmotic pump were implanted into the right proximal tibial physis of twenty six-week-old New Zealand White rabbits. Ten of the pumps were loaded with human recombinant SDF-1alpha, and ten were loaded with phosphate-buffered saline solution (sham treatment). The left leg was used as the uninvolved control. The growth of the tibiae was followed radiographically for eight weeks, and histologic analysis was performed for both the SDF-1-treated rabbits and the sham-treated rabbits at two, four, and eight-week time-points. RESULTS: Radiographic evaluation showed a significant growth inhibition in the SDF-1alpha-treated physes (4.5 +/- 3.0 mm; p = 0.007) compared with the sham-treated physes after eight weeks. No difference was noted when the sham-treated leg was compared with the contralateral, control leg (0.2 +/- 2.9 mm; p = 0.465). Histologic evaluation showed marked physeal disorganization, narrowing, and proteoglycan loss and a significant decrease in physeal height (p < 0.0001) for the SDF-1-treated group. Reversible growth slowing was noted in the uninvolved, control leg of the SDF-1-treated group at six weeks, with resolution of the difference by eight weeks. CONCLUSIONS: SDF-1 may be used to induce physeal closure through a targeted infusion system. However, transient systemic effects of SDF-1 may exist and must be evaluated further prior to its clinical use for epiphysiodesis.

Resorbable fillers reduce stress risers from empty screw holes.

BACKGROUND: Empty screw holes after hardware removal are stress risers that weaken bone and can lead to refracture in an active individual. We sought to reduce these stress risers. We hypothesize that resorbable screws used as hole fillers would (1) provide immediate strength and (2) maintain this strength during resorption. METHODS: Randomized, prospective controlled animal laboratory study with 75 live New Zealand white rabbits' paired femurs. Single mid-diaphyseal holes were filled with a metal or resorbable screw; contralateral femurs were paired empty hole controls. Main outcome measurements included histologic analysis, torsion to failure, peak torque, energy to failure, and stiffness at baseline, 1 week, and 13 weeks postimplantation. RESULTS: At time baseline, resorbable fillers produced an immediate 30% increase in the peak torque (p = 0.01) and 73% increase in peak energy (p = 0.006). Metal screws produced a 17% increase in peak torque (p = 0.038), and a 58% increase in the amount of energy to failure (p = 0.009). At 1 week, although the resorbable (p = 0.01) but not the metal (p = 0.82) screws increased the peak torque, both metal (p = 0.003) and resorbable (p = 0.050) screws increased the peak energy compared with contralateral empty controls. At 13 weeks, metal and resorbable screw-filled bones were as strong as the healed contralateral femurs. Partial screw resorption and new bone formation without lysis was demonstrated histologically. Resorbable screw hole fillers immediately increase the strength of bones without weakening during early resorption. CONCLUSIONS: Placing resorbable fillers in bone defects after hardware removal could reduce the likelihood of refracture.

rhBMP-6 stimulated osteoprogenitor cells enhance posterolateral spinal fusion in the New Zealand white rabbit.

BACKGROUND CONTEXT: The nonunion rate after posterolateral spinal fusion can be as high as 35%. This has stimulated interest in the development of techniques for enhancing new bone formation, including the addition of bioactive peptides or the use of cell-based therapies, including genetically modified cells. In previous studies we have demonstrated that exposing autologous, marrow-derived osteoprogenitor cells to a recombinant human bone morphogenetic protein-6 (rhBMP-6) containing extracellular matrix induces osteoblastic differentiation, and that these cells are capable of increasing new bone formation. Growth of autologous cells on a synthetic rhBMP-6 containing matrix yields a population of stimulated osteoprogenitor cells, without the expense of adding large amounts of rhBMP-6 directly, or the risks inherent in the use of genetically altered cells. PURPOSE: This study was performed to evaluate the potential of rhBMP-6 stimulated osteoprogenitor cells (stOPC) to enhance the rate and strength of posterolateral spinal fusion. STUDY DESIGN: Prospective in vivo animal study OUTCOME MEASURES: Radiographic evidence of spinal fusion, biomechanical testing of explanted spines, histological analysis of new bone formation METHODS: Single-level posterolateral spinal arthrodeses were performed in 69 New Zealand white rabbits. Autologous marrow stem cells were concentrated and then plated on an rhBMP-6-rich extracellular matrix synthesized by genetically engineered mouse C3H10T1/2 cells. Animals in Groups I (n=18) and II (n=18) received autografts of 30M and 60M rhBMP-6 stOPCs in guanidine extracted demineralized bone matrix (gDBM), respectively, whereas those in Group III (n=13) received iliac crest bone graft (ICBG). Those in Group IV (n=10) received gDBM, and those in Group V (n=10) underwent decortication only. Assessment of fusion was made with serial radiographs, manual palpation of the explanted spines, and biomechanical testing. The fusion masses from two animals each in Groups I, II, and IV were evaluated histologically. RESULTS: Fifty-three animals were available for analysis at the conclusion of the study. In these animals, the arthrodesis rate was significantly higher after treatment with rhBMP-6 stOPCs (77% for both Groups I and II by palpation) than ICBG, gDBM, or decortication alone (Group III=55%, IV=20% and V=0%, respectively). Similarly, the peak loads to failure of the fusion masses in Groups I and II (212.5+/-37.8 N and 234.6+/-45.7 N) were significantly greater than the corresponding values in the other groups (Group III=155.9+/-36.4N, Group IV=132.7+/-59.9N, and Group V=92.8+/-18.4N), though when only the fused specimens in Groups I, II, and III were compared, only Group II was significantly different than Group III (234.6+/-45.7N and 155.9+/-36.4N, respectively). The fusion masses in the rhBMP-6 stOPC-treated animals were typified by a thin, fusiform cortical shell, newly formed trabecular bone emanating from the decorticated transverse processes, and residual unremodeled gDBM carrier particles. The fusion masses in the gDBM treated bones were morphologically similar, though they contained less newly formed bone. CONCLUSIONS: The use of rhBMP-6 stOPCs in a carrier of gDBM significantly enhanced the rate and strength of single-level posterolateral spinal arthrodeses in the New Zealand white rabbit, compared with ICBG, gDBM, and decortication alone. Our results confirm that the stimulation of marrow-derived osteoprogenitor cells by growing them on a rhBMP-6 containing extracellular matrix is feasible. Further investigation is warranted to determine the relative contribution of rhBMP-6 stimulation and the number of cells implanted, as well as strategies for optimizing the technique for clinical application.

SHP2 Regulates the Osteogenic Fate of Growth Plate Hypertrophic Chondrocytes.

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.

Horizons in prosthesis development for the restoration of limb function.

The focus of our research program is the restoration of limb function through a biohybrid approach. We consider the limb conceptually as a biohybrid organ consisting of biological tissue, endoprostheses (including neural devices and joint replacements), and exoprostheses. The biohybrid limb maximizes biological function and functional articulations with optimized human-prosthesis interfaces. Our long-term goals are to create biomimetic prostheses, optimized control systems for prostheses, and optimized human-prosthesis interfaces using both limb lengthening and osseointegration techniques.

Effects of Tranexamic Acid Cytotoxicity on In Vitro Chondrocytes.

Use of topical tranexamic acid (TXA) in orthopedic surgery has been expanding over the past decade, with increasing evidence confirming reductions in perioperative blood loss and transfusion requirements, but there is minimal evidence regarding effects of TXA on native cartilage. We conducted a study to understand the in vitro effects of TXA on bovine cartilage and murine chondrocytes and ultimately to expand the clinical application of topical TXA to include scenarios with retained native cartilage, such as hemiarthroplasty. Bovine cartilage explants were exposed to TXA at a concentration of 100 mg/mL, and glycosaminoglycan (GAG) release and cell viability were measured at 8, 24, and 48 hours. Monolayer murine chondrocytes were exposed to TXA 25, 50, and 100 mg/mL, and viability was measured at 8, 24, and 48 hours. GAG released from bovine explants was significantly higher in the samples exposed to TXA 100 mg/mL at all time points. Cell viability was significantly decreased in the explants exposed to TXA 24 and 48 hours after initial incubation. Bovine chondrocyte viability was not affected by TXA 25 mg/mL. Murine chondrocyte viability was similar between the TXA 25 mg/mL and control samples at all time points. The TXA 50 mg/mL sample dropped from 66.51% viability at 8 hours to 6.81% viability at 24 hours and complete cell death by 48 hours. The TXA 100 mg/mL samples had no observable viable cells at 8, 24, and 48 hours. Our data indicated that TXA 100 mg/mL damaged and was cytotoxic to bovine explanted cartilage and was cytotoxic to murine chondrocytes. Murine and bovine chondrocyte viability were not affected by TXA 25 mg/mL.

Identification of the metalloproteinase stromelysin in the physis.

For long bone growth to occur, calcification of the matrix must commence in the lower hypertrophic zone of the growth plate. It is generally accepted that physeal proteoglycans help regulate mineralization, and that at least in vitro, intact proteoglycans can inhibit mineralization. Thus degradation of proteoglycan may be a necessary step prior to calcification. Previous work in our laboratory has demonstrated the presence of neutral metallo-proteases in the growth plate with highest levels in the hypertrophic zone, where calcification occurs. Stromelysin (MMP-3) is a connective tissue matrix-degrading enzyme. It was formerly known as proteoglycanase and is generally considered to be one of the major proteoglycan degrading enzymes in cartilage. Stromelysin is implicated in cartilage destruction in osteoarthritis and may also be involved in tissue remodeling in the physis. Our goal was to determine if the neutral protease previously reported by the authors in the physis was stromelysin. In this study we used Western blots and antibodies to stromelysin and to the stromelysin cleavage site in aggrecan, the most common form of proteoglycan, to demonstrate the presence of stromelysin in the bovine physis. When an antibody raised against the stromelysin cleavage site of aggrecan (FVDIPEN) was incubated with a Western blot, which had been run with aggrecan extracted from bovine physes, a positive reaction resulted. This suggests that there is stromelysin degradation in vivo in the physis. Two different polyclonal antibodies to stromelysin gave positive results on Western blots of purified media from growth plate cultures indicating that stromelysin is produced in vitro in culture. These antibodies also reacted with active stromelysin. The presence of stromelysin in the physis implicates it in physeal physiology. The concentration of its activity in the lower hypertrophic zone and zone of provisional calcification suggests that it may be particularly important in mineralization.

Physiologic weight-bearing increases new vessel formation during distraction osteogenesis: a micro-tomographic imaging study.

During distraction osteogenesis, large volumes of new bone are formed through the slow distraction of fracture callus. The newly formed bone is closely linked to angiogenesis and positively influenced by physiologic loading. In this study, a rat model was used to explore the correlation between these two observations. Unilateral femoral lengthenings were performed in 18 male Sprague-Dawley rats (400-500 g, age<6 months). Half of the animals were allowed to bear weight freely (WB) while the remaining animals were made non-weight-bearing via a through-knee amputation (NWB). After a seven-day latency period, femurs were lengthened 7 mm over 21 days. Animals were sacrificed at 7, 21, 35, and 49 days (0, 4.7, 7, and 7 mm of distraction) at which time lower extremity vessels were perfused with a 60% (w/v) barium sulfate suspension. High-resolution three-dimensional images of the vascular architecture were generated using a fan-beam micro-tomography machine by digitally separating the contrast-filled vessels from surrounding tissue. For two subvolumes, VOI(1), which included vessels in the medullary canal, cortex, and periosteum, and VOI(2), which included only vessels in the canal and cortex, the total tissue volume (TV), the volume of vessels (VV), and vessel diameter (VD) were determined. For the larger subvolume (VOI(1)), VV and vessel density (VV/TV) increased as a function of time (p<0.001) in WB animals. In NWB animals, VV increased significantly with time (p=0.029), while VV/TV did not (p=0.36). Increases in VV and VV/TV were significantly greater in WB animals than in NWB animals (p<0.01 and 0.05, respectively). VD was similar in both groups and did not change with time. Our data suggest that weight bearing stimulates new vessel formation during distraction osteogenesis.

Preformed grafts of porcine small intestine submucosa (SIS) for bridging segmental bone defects.

Previous studies suggest that fresh, morselized porcine small intestine submucosa (SIS) may have promise in the treatment of large bone defects. This study evaluated the bone regenerative potential of preformed tubular SIS grafts, designed to provide a scaffold for regeneration of diaphyseal bone. Critical length segmental defects in the femurs of male rats were either left unfilled (n = 11) or filled with morselized cancellous bone (n = 12), or spanned with intramedullary tubes (n = 12) or periosteal sleeves (n = 12) fabricated from SIS. All of the animals were euthanized 12 weeks postoperatively. Healing was assessed with biweekly radiographs, routine histology, and mechanical testing. Copious new bone formed in the defects of all of the animals treated with cancellous bone; 10 of the 12 animals in that group had healed their defects. In contrast, no new bone was formed in the defects left unfilled or treated with SIS; only fibrous tissue was found. In both of the SIS-treated groups, the SIS persisted at twelve weeks. The cellular response to the SIS involved a mild mononuclear infiltrate in the loose or delaminated superficial layers of the tubes and sleeves, with few cells in the deeper layers. The results of this study cast doubt on the ability of SIS to support or stimulate growth of bone across a critical length segmental bone defect. Additional work will be required to determine whether our results reflect the protocols used to prepare and fabricate the SIS grafts used in the study or the inherent inability of SIS to support new bone growth.

Knee laxity does not vary with the menstrual cycle, before or after exercise.

BACKGROUND: An intriguing explanation for the disproportionately high rate of anterior cruciate ligament injury in female athletes is that the structural properties of the anterior cruciate ligament are affected by the menstrual hormones. Whether this actually occurs, however, is the subject of ongoing debate. HYPOTHESES: (1) Anterior cruciate ligament laxity is different in the follicular, ovulatory, and luteal phases of the menstrual cycle, and (2) exercise exacerbates the difference in anterior cruciate ligament laxity in the 3 phases. METHODS: Over the course of 10 weeks, repeated knee laxity measurements were taken on 27 high-level female athletes, before and after exercise. Point in the menstrual cycle was determined with charts of waking temperature and menstruation. The independent effects of menstrual phase and exercise were evaluated using generalized estimating equations. RESULTS: Data from 18 participants were included in the final analysis. There were no significant differences in anterior cruciate ligament laxity in any of the 3 menstrual phases, before or after exercise. CONCLUSIONS: Anterior cruciate ligament laxity is not significantly different during the follicular, ovulatory, and luteal phases of the menstrual cycle, and bicycling exercise does not exacerbate or create any differences in anterior cruciate ligament laxity.

The risks of overly effective postoperative epidural analgesia.

Continuous epidural analgesia is frequently used to provide supplemental postoperative pain control. Epidural analgesia has the potential to mask the early symptoms that signal impending complications after even routine surgical procedures. We report a case of sciatic nerve palsy following epidural anesthesia after an uncomplicated leg length correction. Good epidural anesthesia may remove a patient's normal protective sensation, allowing pain and other signs of nerve compression from prolonged unchanged postoperative positioning to go unnoticed. This case highlights the need for heightened awareness of potential neurologic compromise in the setting of epidural analgesia. We recommend closely monitoring the patient's neurologic condition and frequently evaluating the patient's position in bed.

Identification of α2-macroglobulin as a master inhibitor of cartilage-degrading factors that attenuates the progression of posttraumatic osteoarthritis.

OBJECTIVE: To determine if supplemental intraarticular α2-macroglobulin (α2 M) has a chondroprotective effect in a rat model of osteoarthritis (OA). METHODS: Using Western blotting, mass spectrometry, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry, α2 M was identified as a potential therapeutic agent through a comparison of α2 M concentrations in serum, synovial fluid (SF), and cartilage from normal subjects and patients with OA. In cultured chondrocytes, the effects of α2 M 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 α2 M, 3) ACLT and 2 IU/kg injections of α2 M, 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. RESULTS: In both normal subjects and OA patients, α2 M 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, α2 M 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 α2 M reduced the concentration of MMP-13 in SF, had a favorable effect on OA-related gene expression, and attenuated OA progression. CONCLUSION: The plasma protease inhibitor α2 M is not present in sufficient concentrations to inactivate the high concentrations of catabolic factors found in OA SF. Our findings suggest that supplemental intraarticular α2 M provides chondral protection in posttraumatic OA.