VEGFA from osteoblasts is not required for lamellar bone formation following tibial loading.

The relationship between osteogenesis and angiogenesis is complex. Normal bone development requires angiogenesis, mediated by vascular endothelial growth factor A (VEGFA). Studies have demonstrated through systemic inhibition or genetic modification that VEGFA is indispensable for several types of bone repair, presumably via its role in supporting angiogenesis. But a direct role for VEGFA within osteoblasts, in the absence of angiogenesis, has also been suggested. To address the question of whether VEGFA from osteoblasts supports bone formation directly, we applied anabolic loading to induce lamellar bone formation in mice, a process shown to be independent of angiogenesis. We hypothesized that VEGFA from osteoblasts is required for lamellar bone formation. To test this hypothesis, we applied axial tibial compression to inducible Cre/LoxP mice from three lines. Vegfafl/fl mice were crossed with Ubiquitin C (UBC), Osterix (Osx) and Dentin-Matrix Protein 1 (DMP1) Cre-ERT2 mice to target all cells, (pre)osteoblast-lineage cells, and mature osteoblasts and osteocytes, respectively. Genotype effects were determined by comparing control (Vegfafl/fl) and Cre+ (VegfaΔ) mice for each line. At 5 months of age tamoxifen was injected for 5 days followed by a 3-week clearance prior to loading. Female and male mice (N = 100) were loaded for 5 days to peak forces to engender -3100 µÎµ peak compressive strain and processed for dynamic histomorphometry (day 12). Percent MS/BS increased 20-70 % as a result of loading, with no effect of genotype in Osx or Dmp1 lines. In contrast, the UBC groups had a significant decrease in relative periosteal BFR/BS in VegfaΔ vs. Vegfafl/fl mice. The UBC line did not have any cortical bone phenotype in non-loaded femurs. In summary, dynamic histomorphometry data confirmed that tibial loading induces lamellar bone formation. Contrary to our hypothesis, there was no decrease in loading-induced bone formation in the Osx or Dmp1 lines in the absence of VEGFA. There was a decrease in bone formation in the UBC line where all cells were targeted. This result indicates that VEGFA from a non-osteoblast cell source supports loading-induced lamellar bone formation, although osteoblast/osteocyte VEGFA is dispensable. These findings support a paracrine model whereby non-osteoblast VEGFA supports lamellar bone formation, independent of angiogenesis.

KIF26B Silencing Prevents Osseous Transdifferentiation of Progenitor/Stem Cells and Attenuates Ectopic Calcification in a Murine Model.

Ectopic calcification is an osteogenic process that leads to the formation of inappropriate bone within intra-articular soft tissues, often in response to injury or surgery. The molecular mechanisms governing this phenotype have yet to be determined. Using a population genetics approach, we identified an association of the kinesin superfamily member 26b (Kif26b) with injury-induced ectopic calcification through quantitative trait locus analysis of recombinant inbred mouse strains, consistent with a genomewide association study that identified KIF26B as a severity locus for ectopic calcification in patients with hip osteoarthritis. Despite these associations of KIF26B with ectopic calcification, its mechanistic role and functional implications have not yet been fully elucidated. Here, we aim to decipher the functional role of KIF26B in osseous and chondrogenic transdifferentiation of human and murine progenitor/stem cells and in a murine model of non-invasive injury-induced intra-articular ectopic calcification. We found that KIF26B ablation via lentivirus-mediated shRNA significantly arrested osteogenesis of progenitor/stem cells and suppressed the expression of typical osteogenic marker genes. Conversely, KIF26B loss-of-function increased chondrogenesis as demonstrated by enhanced Safranin-O staining and by the elevated expression of chondrogenic marker genes. Furthermore, cell function analysis revealed that KIF26B knockdown significantly decreased cell viability and proliferation and induced cellular apoptosis. Mechanistically, loss of osteogenesis was reverted by the addition of a Wnt agonist, SKL2001, demonstrating a role of KIF26B in canonical Wnt/ß-catenin signaling. Finally, intra-articular delivery of Kif26b shRNA in B6-129SF2/J mice significantly hampered the development of intra-articular ectopic calcification at 8 weeks after injury compared with mice treated with non-target scrambled shRNA. In summary, these observations highlight that KIF26B plays a crucial role in ectopic bone formation by repressing osteogenesis, but not chondrogenesis, potentially via modulating Wnt/ß-catenin signaling. These findings establish KIF26B as a critical determinant of the osteogenic process in pathologic endochondral bone formation and an actionable target for pharmacotherapy to mitigate ectopic calcification (and heterotopic ossification). © 2021 American Society for Bone and Mineral Research (ASBMR).

Interleukin-6 (IL-6) deficiency enhances intramembranous osteogenesis following stress fracture in mice.

Interleukin-6 (IL-6) is highly upregulated in response to skeletal injury, suggesting it plays a role in the inflammatory phase of fracture repair. However, the impact of IL-6 on successful repair remains incompletely defined. Therefore, we investigated the role of IL-6 in two models of fracture repair (full fracture and stress fracture) using 12-week old IL-6 global knockout mice (IL-6 KO) and wild type (WT) littermate controls. Callus morphology and mineral density 14 days after full femur fracture did not differ between IL-6 knockout mice and controls. In contrast, IL-6 KO mice had an enhanced bone response 7 days after ulnar stress fracture compared to WT, with increased total callus volume (p = 0.020) and callus bone volume (p = 0.045). IL-6 KO did not alter the recruitment of immune cells (Gr-1 or F4/80 positive) to the stress fracture callus. IL-6 KO also did not alter the number of osteoclasts in the stress fracture callus. Using RNA-seq, we identified differentially expressed genes in stress fracture vs. contralateral control ulnae, and observed that IL-6 KO resulted in only modest alterations to the gene expression response to stress fracture (SFx). Wnt1 was more highly upregulated in IL-6 KO SFx callus at both day 1 (fold change 12.5 in KO vs. 5.7 in WT) and day 3 (fold change 4.7 in KO vs. 1.9 in WT). Finally, using tibial compression to induce bone formation without bone injury, we found that IL-6 KO directly impacted osteoblast function, increasing the propensity for woven bone formation. In summary, we report that IL-6 knockout enhanced formation of callus and bone following stress fracture injury, likely through direct action on the osteoblast's ability to produce woven bone. This suggests a novel role of IL-6 as a suppressor of intramembranous bone formation.

Aging aggravates intervertebral disc degeneration by regulating transcription factors toward chondrogenesis.

Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of Wnt signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.

Congenital lipodystrophy induces severe osteosclerosis.

Berardinelli-Seip congenital generalized lipodystrophy is associated with increased bone mass suggesting that fat tissue regulates the skeleton. Because there is little mechanistic information regarding this issue, we generated "fat-free" (FF) mice completely lacking visible visceral, subcutaneous and brown fat. Due to robust osteoblastic activity, trabecular and cortical bone volume is markedly enhanced in these animals. FF mice, like Berardinelli-Seip patients, are diabetic but normalization of glucose tolerance and significant reduction in circulating insulin fails to alter their skeletal phenotype. Importantly, the skeletal phenotype of FF mice is completely rescued by transplantation of adipocyte precursors or white or brown fat depots, indicating that adipocyte derived products regulate bone mass. Confirming such is the case, transplantation of fat derived from adiponectin and leptin double knockout mice, unlike that obtained from their WT counterparts, fails to normalize FF bone. These observations suggest a paucity of leptin and adiponectin may contribute to the increased bone mass of Berardinelli-Seip patients.

VEGFA From Early Osteoblast Lineage Cells (Osterix+) Is Required in Mice for Fracture Healing.

Bone formation via intramembranous and endochondral ossification is necessary for successful healing after a wide range of bone injuries. The pleiotropic cytokine, vascular endothelial growth factor A (VEGFA) has been shown, via nonspecific pharmacologic inhibition, to be indispensable for angiogenesis and ossification following bone fracture and cortical defect repair. However, the importance of VEGFA expression by different cell types during bone healing is not well understood. We sought to determine the role of VEGFA from different osteoblast cell subsets following clinically relevant models of bone fracture and cortical defect. Ubiquitin C (UBC), Osterix (Osx), or Dentin matrix protein 1 (Dmp1) Cre-ERT2 mice (male and female) containing floxed VEGFA alleles (VEGFAfl/fl ) were either given a femur full fracture, ulna stress fracture, or tibia cortical defect at 12 weeks of age. All mice received tamoxifen continuously starting 2 weeks before bone injury and throughout healing. UBC Cre-ERT2 VEGFAfl/fl (UBC cKO) mice, which were used to mimic nonspecific inhibition, had minimal bone formation and impaired angiogenesis across all bone injury models. UBC cKO mice also exhibited impaired periosteal cell proliferation during full fracture, but not stress fracture repair. Osx Cre-ERT2 VEGFAfl/fl (Osx cKO) mice, but not Dmp1 Cre-ERT2 VEGFAfl/fl (Dmp1 cKO) mice, showed impaired periosteal bone formation and angiogenesis in models of full fracture and stress fracture. Neither Osx cKO nor Dmp1 cKO mice demonstrated significant impairments in intramedullary bone formation and angiogenesis following cortical defect. These data suggest that VEGFA from early osteolineage cells (Osx+), but not mature osteoblasts/osteocytes (Dmp1+), is critical at the time of bone injury for rapid periosteal angiogenesis and woven bone formation during fracture repair. Whereas VEGFA from another cell source, not from the osteoblast cell lineage, is necessary at the time of injury for maximum cortical defect intramedullary angiogenesis and osteogenesis. © 2019 American Society for Bone and Mineral Research.

Gli1 identifies osteogenic progenitors for bone formation and fracture repair.

Bone formation in mammals requires continuous production of osteoblasts throughout life. A common molecular marker for all osteogenic mesenchymal progenitors has not been identified. Here, by lineage-tracing experiments in fetal or postnatal mice, we discover that Gli1+ cells progressively produce osteoblasts in all skeletal sites. Most notably, in postnatal growing mice, the Gli1+ cells residing immediately beneath the growth plate, termed here "metaphyseal mesenchymal progenitors" (MMPs), are essential for cancellous bone formation. Besides osteoblasts, MMPs also give rise to bone marrow adipocytes and stromal cells in vivo. RNA-seq reveals that MMPs express a number of marker genes previously assigned to mesenchymal stem/progenitor cells, including CD146/Mcam, CD44, CD106/Vcam1, Pdgfra, and Lepr. Genetic disruption of Hh signaling impairs proliferation and osteoblast differentiation of MMPs. Removal of ß-catenin causes MMPs to favor adipogenesis, resulting in osteopenia coupled with increased marrow adiposity. Finally, postnatal Gli1+ cells contribute to both chondrocytes and osteoblasts during bone fracture healing. Thus Gli1 marks mesenchymal progenitors responsible for both normal bone formation and fracture repair.

Therapeutic efficacy of intra-articular hyaluronan derivative and platelet-rich plasma in mice following axial tibial loading.

OBJECTIVE: To investigate the therapeutic potential of intra-articular hyaluronan-derivative HYADD® 4-G and/or platelet-rich plasma (PRP) in a mouse model of non-invasive joint injury. METHODS: Non-invasive axial tibial loading was used to induce joint injury in 10-week-old C57BL/6J mice (n = 86). Mice underwent a single loading of either 6 Newton (N) or 9N axial tibial compression. HYADD® 4-G was injected intra-articularly at 8 mg/mL or 15 mg/mL either before or after loading with or without PRP. Phosphate-buffered-saline was injected as control. Knee joints were harvested at 5 or 56 days post-loading and prepared for micro-computed tomography scanning and subsequently processed for histology. Immunostaining was performed for aggrecan to monitor its distribution, for CD44 to monitor chondrocyte reactive changes and for COMP (cartilage oligomeric matrix protein) as an index for cartilage matrix changes related to loading and cartilage injury. TUNEL assay was performed to identify chondrocyte apoptosis. RESULTS: Loading initiated cartilage proteoglycan loss and chondrocyte apoptosis within 5 days with slowly progressive post-traumatic osteoarthritis (no cartilage degeneration, but increased synovitis and ectopic calcification after 9N loading) at 56 days. Mice treated with repeated HYADD® 4-G (15 mg/mL) or HYADD® 4-G (8 mg/mL) ± PRP or PRP alone exhibited no significant improvement in the short-term (5 days) and long-term (56 days) consequences of joint loading except for a trend for improved bone changes compared to non-loaded joints. CONCLUSION: While we failed to show an overall effect of intra-articular delivery of hyaluronan-derivative and/or PRP in reversing/protecting the pathological events in cartilage and synovium following joint injury, some bone alterations were relatively less severe with hyaluronan-derivative at higher concentration or in association with PRP.

Mechanical comparison of iliosacral reconstruction techniques after sarcoma resection.

BACKGROUND: Reconstruction of iliosacral defects following oncologic resection is a difficult clinical problem associated with a high incidence of failure. Technical approaches to this problem are heterogeneous and evidence supporting specific techniques is sparse. Maximizing construct stability may improve union rates and functional outcomes. The purpose of this study is to compare construct stiffness, load to failure, and mechanism of failure between two methods of iliosacral reconstruction in an ex-vivo model to determine if either is mechanically superior. METHODS: Eight third-generation composite pelves reconstructed with a plate-and-screw technique were tested against seven pelves reconstructed with a minimal spinal instrumentation technique using axial loading in a double-leg stance model. FINDINGS: The pelves from the plate group demonstrated higher stiffness in the direction of applied load (102.9 vs. 66.8N/mm; p=0.010) and endured a significantly larger maximum force (1416 vs. 1059N; p=0.015) than the rod group prior to failure. Subjectively, the rod-reconstructed pelves were noted to be rotationally unconstrained while pivoting around their single point-of fixation in each segment leading to earlier failure. INTERPRETATION: Plate-reconstruction was mechanically superior to spinal instrumentation in the manner performed in this study. More than one point of fixation in each segment should be achieved to minimize the risk of rotational deformation.

Adipose-derived mesenchymal stromal cells modulate tendon fibroblast responses to macrophage-induced inflammation in vitro.

INTRODUCTION: Macrophage-driven inflammation is a key feature of the early period following tendon repair, but excessive inflammation has been associated with poor clinical outcomes. Modulation of the inflammatory environment using molecular or cellular treatments may provide a means to enhance tendon healing. METHODS: To examine the effect of pro-inflammatory cytokines secreted by macrophages on tendon fibroblasts (TF), we established in vitro models of cytokine and macrophage-induced inflammation. Gene expression, protein expression, and cell viability assays were used to examine TF responses. In an effort to reduce the negative effects of inflammatory cytokines on TFs, adipose-derived mesenchymal stromal cells (ASCs) were incorporated into the model and their ability to modulate inflammation was investigated. RESULTS: The inflammatory cytokine interleukin 1 beta (IL-1ß) and macrophages of varying phenotypes induced up-regulation of pro-inflammatory factors and matrix degradation factors and down-regulation of factors related to extracellular matrix formation by TFs in culture. ASCs did not suppress these presumably negative effects induced by IL-1ß. However, ASC co-culture with M1 (pro-inflammatory) macrophages successfully suppressed the effects of M1 macrophages on TFs by inducing a phenotypic switch from a pro-inflammatory macrophage phenotype to an anti-inflammatory macrophage phenotype, thus resulting in exposure of TFs to lower levels of pro-inflammatory cytokines (e.g., IL-1ß, tumor necrosis factor alpha (TNFα)). CONCLUSIONS: These findings suggest that IL-1ß and M1 macrophages are detrimental to tendon healing and that ASC-mediated modulation of the post-operative inflammatory response may be beneficial for tendon healing.

Experimental and computational analysis of composite ankle-foot orthosis.

Carbon fiber (CF) ankle-foot orthoses (AFOs) can improve gait by increasing ankle plantar-flexor power and improving plantar-flexor ankle joint moment and energy efficiency compared with posterior leaf spring AFOs made of thermoplastic. However, fabricating a CF AFO to optimize the performance of the individual user may require multiple AFOs and expensive fabrication costs. Finite element analysis (FEA) models were developed to predict the mechanical behavior of AFOs in this study. Three AFOs, two made of CF composite material and one made of thermoplastic material, were fabricated and then mechanically tested to produce force-displacement data. The FEA models were validated by comparing model predictions with mechanical testing data performed under the same loading and boundary conditions. The actual mechanical testing demonstrated that CF performs better than thermoplastic. The simulation results showed that FEA models produced accurate predictions for both types of orthoses. The relative error of the energy return ratio predicted by the CF AFO FEA model developed in this study is less than 3%. We conclude that highly accurate FEA models will allow orthotists to improve CF AFO fabrication without wasting resources (time and money) on trial and error fabrications that are expensive and do not consistently improve AFO and user performance.

The effect of core and epitendinous suture modifications on repair of intrasynovial flexor tendons in an in vivo canine model.

PURPOSE: To determine in vivo effects of modifications to core and epitendinous suture techniques in a canine intrasynovial flexor tendon repair model using clinically relevant rehabilitation. Our null hypothesis was that gap formation and rupture rates would remain consistent across repair techniques. METHODS: We evaluated gap formation and rupture in 75 adult mongrel dogs that underwent repair of intrasynovial flexor tendon lacerations followed by standardized postoperative therapy. The current suture technique was a 4-0, 8-strand core suture with a purchase of 1.2 cm and a 5-0, epitendinous suture repair with a 2-mm purchase length and depth. We compared gap and failure by chi-square analysis to a historical group of in vivo repairs (n = 76) from the same canine model using 8-strand core suture repair with purchase of 0.75 cm and 6-0 epitendinous suture with a 1-mm purchase length and depth. RESULTS: A total of 93% of tendons (n = 70) demonstrated gapping of less than 3 mm using the current suture technique. Five percent of tendons (n = 4) had a gap of 3 mm or greater, and there was 1 repair site failure. This was significantly improved over the comparison group of historical 8-strand core repair technique, which resulted in 82% (n = 62) of repairs with a gap of less than 3 mm and 7 failures (9%). CONCLUSIONS: In an in vivo model, current modifications to suture techniques for intrasynovial flexor tendon repair demonstrated significant improvements in gap formation and rupture compared with a similar technique using shorter purchase lengths and shallower purchase depth. CLINICAL RELEVANCE: Suggested repair modifications for the treatment of zone II flexor tendon transections demonstrate improvements in gap formation and tendon rupture in vivo.

Effect of combined traumatic impact and radial transection of medial meniscus on knee articular cartilage in a rabbit in vivo model.

PURPOSE: The purpose of this study was to test the hypothesis that combined meniscectomy and traumatic impact accelerate early degeneration of articular cartilage in the knee versus meniscectomy alone. METHODS: A previously published in vivo rabbit cartilage impact model was used combined with radial transection of the medial meniscus posterior horn versus meniscal transection alone. Rabbits were killed 3 months after surgery. Quantitative histologic analysis of the articular cartilage proteoglycan depth and glycosaminoglycan (GAG) fraction was performed at the site of impact on the posterior femoral condyle (PFC) and at the distal femoral condyle (DFC) overlying the meniscectomy in the surgical knee and the contralateral control knee. RESULTS: The articular cartilage in the knees that underwent isolated meniscectomy did not differ significantly from the contralateral control knees for any measured value. The knees with a combined insult had a lower GAG fraction (P = .03) at the PFC and a greater depth of proteoglycan loss at both the PFC (P = .02) and the DFC (P = .04) versus contralateral controls. Compared with meniscectomy alone, the combined-insult knees had a greater depth of proteoglycan loss at the DFC (P = .005). CONCLUSIONS: On the basis of early results using GAG fraction and proteoglycan depth, combined traumatic impact and meniscectomy are more damaging to articular cartilage than meniscectomy alone. CLINICAL RELEVANCE: A knee with a combination of meniscal injury and articular cartilage impact may be at particularly high risk for early joint degeneration.

Effect of bone morphogenetic protein 2 on tendon-to-bone healing in a canine flexor tendon model.

Tendon-to-bone healing is typically poor, with a high rate of repair-site rupture. Bone loss after tendon-to-bone repair may contribute to poor outcomes. Therefore, we hypothesized that the local application of the osteogenic growth factor bone morphogenetic protein 2 (BMP-2) would promote bone formation, leading to improved repair-site mechanical properties. Intrasynovial canine flexor tendons were injured in Zone 1 and repaired into bone tunnels in the distal phalanx. BMP-2 was delivered to the repair site using either a calcium phosphate matrix (CPM) or a collagen sponge (COL) carrier. Each animal also received carrier alone in an adjacent repair to serve as an internal control. Repairs were evaluated at 21 days using biomechanical, radiographic, and histologic assays. Although an increase in osteoid formation was noted histologically, no significant increases in bone mineral density occurred. When excluding functional failures (i.e., ruptured and gapped repairs), mechanical properties were not different when comparing BMP-2/CPM groups with carrier controls. A significantly higher percentage of BMP-2 treated specimens had a maximum force <20 N compared to carrier controls. While tendon-to-bone healing can be enhanced by addressing the bone loss that typically occurs after surgical repair, the delivery of BMP-2 using the concentrations and methods of the current study did not improve mechanical properties over carrier alone. The anticipated anabolic effect of BMP-2 was insufficient in the short time frame of this study to counter the post-repair loss of bone.

Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice.

There are conflicting data on whether age reduces the response of the skeleton to mechanical stimuli. We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months (p<0.001). There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months (p<0.001). We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 µÎµ endocortical; -2350 µÎµ periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls (p = 0.001). In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias (p<0.05), due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%; p<0.05 vs. other ages). In summary, non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice. We conclude that mechanical loading of the tibia is anabolic for cortical bone in young and middle-aged female BALB/c mice.

The effects of screw length on stability of simulated osteoporotic distal radius fractures fixed with volar locking plates.

PURPOSE: Volar plating for distal radius fractures has caused extensor tendon ruptures resulting from dorsal screw prominence. This study was designed to determine the biomechanical impact of placing unicortical distal locking screws and pegs in an extra-articular fracture model. METHODS: We applied volar-locking distal radius plates to 30 osteoporotic distal radius models. We divided radiuses into 5 groups based on distal locking fixation: bicortical locked screws, 3 lengths of unicortical locked screws (abutting the dorsal cortex [full length], 75% length, and 50% length to dorsal cortex), and unicortical locked pegs. Distal radius osteotomy simulated a dorsally comminuted, extra-articular fracture. We determined each construct's stiffness under physiologic loads (axial compression, dorsal bending, and volar bending) before and after 1,000 cycles of axial conditioning and before axial loading to failure (2 mm of displacement) and subsequent catastrophic failure. RESULTS: Cyclic conditioning did not alter the constructs' stiffness. Stiffness to volar bending and dorsal bending forces were similar between groups. Final stiffness under axial load was statistically equivalent for all groups: bicortical screws (230 N/mm), full-length unicortical screws (227 N/mm), 75% length unicortical screws (226 N/mm), 50% length unicortical screws (187 N/mm), and unicortical pegs (226 N/mm). Force at 2-mm displacement was significantly less for 50% length unicortical screws (311 N) compared with bicortical screws (460 N), full-length unicortical screws (464 N), 75% length unicortical screws (400 N), and unicortical pegs (356 N). Force to catastrophic fracture was statistically equivalent between groups, but mean values for pegs (749 N) and 50% length unicortical (702 N) screws were 16% to 21% less than means for bicortical (892 N), full-length unicortical (860 N), and 75% length (894 N) unicortical constructs. CONCLUSIONS: Locked unicortical distal screws of at least 75% length produce construct stiffness similar to bicortical fixation. Unicortical distal fixation for extra-articular distal radius fractures should be entertained to avoid extensor tendon injury because this technique does not appear to compromise initial fixation. CLINICAL RELEVANCE: Using unicortical fixation during volar distal radius plating may protect extensor tendons without compromising fixation.

The effects of exogenous basic fibroblast growth factor on intrasynovial flexor tendon healing in a canine model.

BACKGROUND: Studies have demonstrated that flexor tendon repair strength fails to increase in the first three weeks following suturing of the tendon, a finding that correlates closely with the timing of many clinical failures. The application of growth factors holds promise for improving the tendon-repair response and obviating failure in the initial three weeks. METHODS: The effects of basic fibroblast growth factor on flexor tendon healing were evaluated with use of a canine model. Operative repair followed by the sustained delivery of basic fibroblast growth factor, at two different doses, was compared with operative repair alone. Histological, biochemical, and biomechanical methods were used to evaluate the tendons twenty-one days after repair. RESULTS: Vascularity, cellularity, and adhesion formation were increased in the tendons that received basic fibroblast growth factor as compared with the tendons that received operative repair alone. DNA concentration was increased in the tendons that received 1000 ng of basic fibroblast growth factor (mean and standard deviation, 5.7 ± 0.7 µg/mg) as compared with the tendons that received 500 ng of basic fibroblast growth factor (3.8 ± 0.7 µg/mg) and the matched control tendons that received operative repair alone (4.5 ± 0.9 µg/mg). Tendons that were treated with basic fibroblast growth factor had a lower ratio of type-I collagen to type-III collagen, indicating increased scar formation compared with that seen in tendons that received operative repair alone (3.0 ± 1.6 in the group that received 500-ng basic fibroblast growth factor compared with 4.3 ± 1.0 in the paired control group that received operative repair alone, and 3.4 ± 0.6 in the group that received 1000-ng basic fibroblast growth factor compared with 4.5 ± 1.9 in the paired control group that received operative repair alone). Consistent with the increases in adhesion formation that were seen in tendons treated with basic fibroblast growth factor, the range of motion was reduced in the group that received the higher dose of basic fibroblast growth factor than it was in the paired control group that received operative repair alone (16.6° ± 9.4° in the group that received 500 ng basic fibroblast growth factor, 13.4° ± 6.1° in the paired control group that received operative repair alone, and 29.2° ± 5.8° in the normal group [i.e., the group of corresponding, uninjured tendons from the contralateral forelimb]; and 15.0° ± 3.8° in the group that received 1000 ng basic fibroblast growth factor, 19.3° ± 5.5° in the paired control group that received operative repair alone, and 29.0° ± 8.8° in the normal group). There were no significant differences in tendon excursion or tensile mechanical properties between the groups that were treated with basic fibroblast growth factor and the groups that received operative repair alone. CONCLUSIONS: Although basic fibroblast growth factor accelerated the cell-proliferation phase of tendon healing, it also promoted neovascularization and inflammation in the earliest stages following the suturing of the tendon. Despite a substantial biologic response, the administration of basic fibroblast growth factor failed to produce improvements in either the mechanical or functional properties of the repair. Rather, increased cellular activity resulted in peritendinous scar formation and diminished range of motion.

Technical and biological modifications for enhanced flexor tendon repair.

Clinical outcomes after intrasynovial flexor tendon repair have been substantially improved over the past 2 decades through advances in tendon suture techniques and postoperative rehabilitation methods. Nevertheless, complications such as repair site elongation (i.e., gap formation) and rupture continue to occur frequently. Experimental studies have shown that repair site strength fails to increase in the first 3 weeks after tendon suture. After 3 weeks, the strength and rigidity of the repair site improve significantly, a process that continues for several months. Formation of a repair site gap during the early rehabilitation period has been shown to considerably delay the accrual of repair site strength over time. Thus, it is of prime importance that the method of tendon suture achieves and maintains a stiff and strong repair site during the early healing interval by maintaining close approximation of the tendon stumps and by stimulating, where possible, the intrinsic repair response. In this review, we describe recent efforts to enhance the integrity of the immature repair site. We focus on 2 major areas of advancement: surgical technique modifications and manipulation of the biologic and biochemical environment.

Microfibril-associated glycoprotein-1, an extracellular matrix regulator of bone remodeling.

MAGP1 is an extracellular matrix protein that, in vertebrates, is a ubiquitous component of fibrillin-rich microfibrils. We previously reported that aged MAGP1-deficient mice (MAGP1Delta) develop lesions that are the consequence of spontaneous bone fracture. We now present a more defined bone phenotype found in MAGP1Delta mice. A longitudinal DEXA study demonstrated age-associated osteopenia in MAGP1Delta animals and muCT confirmed reduced bone mineral density in the trabecular and cortical bone. Further, MAGP1Delta mice have significantly less trabecular bone, the trabecular microarchitecture is more fragmented, and the diaphyseal cross-sectional area is significantly reduced. The remodeling defect seen in MAGP1Delta mice is likely not due to an osteoblast defect, because MAGP1Delta bone marrow stromal cells undergo osteoblastogenesis and form mineralized nodules. In vivo, MAGP1Delta mice exhibit normal osteoblast number, mineralized bone surface, and bone formation rate. Instead, our findings suggest increased bone resorption is responsible for the osteopenia. The number of osteoclasts derived from MAGP1Delta bone marrow macrophage cells is increased relative to the wild type, and osteoclast differentiation markers are expressed at earlier time points in MAGP1Delta cells. In vivo, MAGP1Delta mice have more osteoclasts lining the bone surface. RANKL (receptor activator of NF-kappaB ligand) expression is significantly higher in MAGP1Delta bone, and likely contributes to enhanced osteoclastogenesis. However, bone marrow macrophage cells from MAGP1Delta mice show a higher propensity than do wild-type cells to differentiate to osteoclasts in response to RANKL, suggesting that they are also primed to respond to osteoclast-promoting signals. Together, our findings suggest that MAGP1 is a regulator of bone remodeling, and its absence results in osteopenia associated with an increase in osteoclast number.

bFGF and PDGF-BB for tendon repair: controlled release and biologic activity by tendon fibroblasts in vitro.

Flexor tendon injuries are often encountered clinically and typically require surgical repair. Return of function after repair is limited due to adhesion formation, which leads to reduced tendon gliding, and due to a lack of repair site strength, which leads to repair site gap formation or rupture. The application of the growth factors basic fibroblastic growth factor (bFGF) and platelet derived growth factor BB (PDGF-BB) has been shown to have the potential to enhance tendon healing. The objectives of this study were to examine: (1) the conditions over which delivery of bFGF can be controlled from a heparin-binding delivery system (HBDS) and (2) the effect of bFGF and PDGF-BB released from this system on tendon fibroblast proliferation and matrix gene expression in vitro over a 10-day interval. Delivery of bFGF was controlled using a HBDS. Fibrin matrices containing the HBDS retained bFGF better than did matrices lacking the delivery system over the 10-day period studied. Delivery of bFGF and PDGF-BB using the HBDS stimulated tendon fibroblast proliferation and promoted changes in the expression of matrix genes related to tendon gliding, strength, and remodeling. Both growth factors may be effective in enhancing tendon healing in vivo.