Multi-organ phenotypes in mice lacking latent TGFß binding protein 2 (LTBP2).

BACKGROUND: Latent TGFß binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFß, although it may interfere with the function of other LTBPs or interact with other signaling pathways. RESULTS: Here, we investigate mice lacking Ltbp2 (Ltbp2-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype. CONCLUSIONS: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.

Loading-induced bone formation is mediated by Wnt1 induction in osteoblast-lineage cells.

Mechanical loading on the skeleton stimulates bone formation. Although the exact mechanism underlying this process remains unknown, a growing body of evidence indicates that the Wnt signaling pathway is necessary for the skeletal response to loading. Recently, we showed that Wnts produced by osteoblast lineage cells mediate the osteo-anabolic response to tibial loading in adult mice. Here, we report that Wnt1 specifically plays a crucial role in mediating the mechano-adaptive response to loading. Independent of loading, short-term loss of Wnt1 in the Osx-lineage resulted in a decreased cortical bone area in the tibias of 5-month-old mice. In females, strain-matched loading enhanced periosteal bone formation in Wnt1F/F controls, but not in Wnt1F/F; OsxCreERT2 knockouts. In males, strain-matched loading increased periosteal bone formation in both control and knockout mice; however, the periosteal relative bone formation rate was 65% lower in Wnt1 knockouts versus controls. Together, these findings show that Wnt1 supports adult bone homeostasis and mediates the bone anabolic response to mechanical loading.

Cyclic testing of six-strand suture techniques for zone 2 flexor tendon lacerations.

BACKGROUND: Biomechanical analysis using cyclic testing for repaired flexor tendons is a clinically relevant method. The aim of this study was to evaluate the tensile properties of two six-strand suture techniques, the triple looped suture and Yoshizu #1 suture techniques using cyclic testing under simulating early active mobilization conditions. METHODS: Twenty-five flexor digitorum profundus tendons harvested from fresh frozen human cadaver hands were repaired in zone 2 utilizing one of three repair techniques: the 2-strand modified Kessler (MK) technique as a control, the triple looped suture (TLS) and Yoshizu #1 suture (Y1) techniques. In each suture technique, 4-0 monofilament nylon sutures were used for core sutures and 6-0 monofilament nylon sutures for circumferential running sutures. Cyclic testing was performed using 20 N with 600 cycles at 1 Hz. RESULTS: Five out of eight specimens in the MK group ruptured during cyclic testing. Thus, this group was excluded from analysis. On the other hand, all tendons in the TLS and Y1 groups tolerated cyclic testing. Average gaps of the TLS and Y1 groups were 0.5 ± 0.8 mm and 1.9 ± 2.2 mm, respectively. All tendons in the TLS group and six out of nine tendons in the Y1 group formed gaps less than 2 mm. Two tendons in the Y1 group formed a gap of 3.8 and 6.6 mm had breakage of peripheral sutures at the first cycle. Mean ultimate tensile force of the TLS and Y1 group measured after cyclic tensing, were 66.2 ± 9.0 N and 65.9 ± 13.1 N, respectively. No statistical difference between the two groups was found in gap and ultimate tensile forces. CONCLUSIONS: This study suggested that the TLS and Y1 techniques have tensile properties to allow early active mobilization. None of tendons repaired with the TLS technique had gaps more than 2 mm.

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.

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.

Proliferating osteoblasts are necessary for maximal bone anabolic response to loading in mice.

Following mechanical loading, osteoblasts may arise via activation, differentiation, or proliferation to form bone. Our objective was to ablate proliferating osteoblast lineage cells in order to investigate the importance of these cells as a source for loading-induced bone formation. We utilized 3.6Col1a1-tk mice in which replicating osteoblast lineage cells can be ablated in an inducible manner using ganciclovir (GCV). Male and female mice were aged to 5- and 12-months and subjected to 5 days of tibial compression. "Experimental" mice were tk-positive, treated with GCV; "control" mice were either tk-negative treated with GCV, or tk-positive treated with PBS. We confirmed that experimental mice had a decrease in tk-positive cells that arose from proliferation. Next, we assessed bone formation after loading to low (7N) and high (11N) forces and observed that periosteal bone formation rate in experimental mice was reduced by approximately 70% for both forces. Remarkably, woven bone formation induced by high-force loading was blocked in experimental mice. Loading-induced lamellar bone formation was diminished but not prevented in experimental mice. We conclude that osteoblast proliferation induced by mechanical loading is a critical source of bone forming osteoblasts for maximal lamellar formation and is essential for woven bone formation.

Mechanosensitive Ca2+ signaling and coordination is diminished in osteocytes of aged mice during ex vivo tibial loading.

Purpose: The osteocyte is considered the major mechanosensor in bone, capable of detecting forces at a cellular level to coordinate bone formation and resorption. The pathology of age-related bone loss, a hallmark of osteoporosis, is attributed in part to impaired osteocyte mechanosensing. However, real-time evidence of the effect of aging on osteocyte responses to mechanical load is lacking. Intracellular calcium (Ca2+) oscillations have been characterized as an early mechanosensitive response in osteocytes in systems of multiple scales and thus can serve as a real-time measure of osteocyte mechanosensitivity. Our objective was to utilize an ex vivo model to investigate potentially altered mechanosensing in the osteocyte network with aging.Methods: Tibiae were explanted from young-adult (5 mo) and aged (22 mo) female mice and incubated with Fluo-8 AM to visualize osteocyte intracellular Ca2+. Whole tibiae were cyclically loaded while in situ osteocyte Ca2+ dynamics were simultaneously imaged with confocal microscopy. Responsive osteocyte percentage and Ca2+ peak characteristics were quantified, as well as signaling synchrony between paired cells in the field of view.Results: Fewer osteocytes responded to mechanical loading in aged mice compared to young-adult and did so in a delayed manner. Osteocytes from aged mice also lacked the well-correlated relationship between Ca2+ signaling synchrony and cell-cell distance exhibited by young-adult osteocytes.Conclusions: We have demonstrated, for the first time, real-time evidence of the diminished mechanosensing and lack of signaling coordination in aged osteocyte networks in tibial explants, which may contribute to pathology of age-induced bone loss.

Loss of scleraxis in mice leads to geometric and structural changes in cortical bone, as well as asymmetry in fracture healing.

Scleraxis (Scx) is a known regulator of tendon development, and recent work has identified the role of Scx in bone modeling. However, the role of Scx in fracture healing has not yet been explored. This study was conducted to identify the role of Scx in cortical bone development and fracture healing. Scx green fluorescent protein-labeled (ScxGFP) reporter and Scx-knockout (Scx-mutant) mice were used to assess bone morphometry and the effects of fracture healing on Scx localization and gene expression, as well as callus healing response. Botulinum toxin (BTX) was used to investigate muscle unloading effects on callus shape. Scx-mutant long bones had structural and mechanical defects. Scx gene expression was elevated and bmp4 was decreased at 24 h after fracture. ScxGFP+ cells were localized throughout the healing callus after fracture. Scx-mutant mice demonstrated disrupted callus healing and asymmetry. Asymmetry of Scx-mutant callus was not due to muscle unloading. Wild-type littermates (age matched) served as controls. This is the first study to explore the role of Scx in cortical bone mechanics and fracture healing. Deletion of Scx during development led to altered long bone properties and callus healing. This study also demonstrated that Scx may play a role in the periosteal response during fracture healing.-McKenzie, J. A., Buettmann, E., Abraham, A. C., Gardner, M. J., Silva, M. J., Killian, M. L. Loss of scleraxis in mice leads to geometric and structural changes in cortical bone, as well as asymmetry in fracture healing.

Spatial histomorphometry reveals that local peripheral nerves modulate but are not required for skeletal adaptation to applied load in mice.

Mechanical loading is required for bone health and results in skeletal adaptation to optimize strength. Local nerve axons, particularly within the periosteum, may respond to load-induced biomechanical and biochemical cues. However, their role in the bone anabolic response remains controversial. We hypothesized that spatial alignment of periosteal nerves with sites of load-induced bone formation would clarify this relationship. To achieve this, we developed RadialQuant, a custom tool for spatial histomorphometry. Tibiae of control and neurectomized (sciatic/femoral nerve cut) pan-neuronal Baf53b-tdTomato reporter mice were loaded for 5-days. Bone formation and periosteal nerve axon density were then quantified simultaneously in non-decalcified sections of the mid-diaphysis using RadialQuant. In control animals, anabolic loading induced maximal periosteal bone formation at the site of peak compression, as has been reported previously. Loading did not significantly change overall periosteal nerve density. However, a trending 28% increase in periosteal axons was noted at the site of peak compression in loaded limbs. Neurectomy depleted 88% of all periosteal axons, with near-total depletion on load-responsive surfaces. Neurectomy alone also caused de novo bone formation on the lateral aspect of the mid-diaphysis. However, neurectomy did not inhibit load-induced increases in periosteal bone area, mineralizing surface, or bone formation rate. Rather, neurectomy spatially redistributed load-induced bone formation towards the lateral tibial surface with a reduction in periosteal bone formation at the posterolateral apex (-63%) and enhancement at the lateral surface (+1360%). Altogether, this contributed to comparable load-induced changes in cortical bone area fraction (+4.4% in controls; +5.4% in neurectomized). Our results show that local skeletal innervation modulates but is not required for skeletal adaptation to applied load. This supports the continued use of loading and weight-bearing exercise as an effective strategy to increase bone mass, even in patients with peripheral nerve damage or dysfunction.

Multi-scale cortical bone traits vary in females and males from two mouse models of genetic diversity.

Understanding the genetic basis of cortical bone traits can allow for the discovery of novel genes or biological pathways regulating bone health. Mice are the most widely used mammalian model for skeletal biology and allow for the quantification of traits that cannot easily be evaluated in humans, such as osteocyte lacunar morphology. The goal of our study was to investigate the effect of genetic diversity on multi-scale cortical bone traits of 3 long bones in skeletally-mature mice. We measured bone morphology, mechanical properties, material properties, lacunar morphology, and mineral composition of mouse bones from 2 populations of genetic diversity. Additionally, we compared how intrabone relationships varied in the 2 populations. Our first population of genetic diversity included 72 females and 72 males from the 8 inbred founder strains used to create the Diversity Outbred (DO) population. These 8 strains together span almost 90% of the genetic diversity found in mice (Mus musculus). Our second population of genetic diversity included 25 genetically unique, outbred females and 25 males from the DO population. We show that multi-scale cortical bone traits vary significantly with genetic background; heritability values range from 21% to 99% indicating genetic control of bone traits across length scales. We show for the first time that lacunar shape and number are highly heritable. Comparing the 2 populations of genetic diversity, we show that each DO mouse does not resemble a single inbred founder, but instead the outbred mice display hybrid phenotypes with the elimination of extreme values. Additionally, intrabone relationships (eg, ultimate force vs. cortical area) were mainly conserved in our 2 populations. Overall, this work supports future use of these genetically diverse populations to discover novel genes contributing to cortical bone traits, especially at the lacunar length scale.

Pitx1 haploinsufficiency causes clubfoot in humans and a clubfoot-like phenotype in mice.

Clubfoot affects 1 in 1000 live births, although little is known about its genetic or developmental basis. We recently identified a missense mutation in the PITX1 bicoid homeodomain transcription factor in a family with a spectrum of lower extremity abnormalities, including clubfoot. Because the E130K mutation reduced PITX1 activity, we hypothesized that PITX1 haploinsufficiency could also cause clubfoot. Using copy number analysis, we identified a 241 kb chromosome 5q31 microdeletion involving PITX1 in a patient with isolated familial clubfoot. The PITX1 deletion segregated with autosomal dominant clubfoot over three generations. To study the role of PITX1 haploinsufficiency in clubfoot pathogenesis, we began to breed Pitx1 knockout mice. Although Pitx1(+/-) mice were previously reported to be normal, clubfoot was observed in 20 of 225 Pitx1(+/-) mice, resulting in an 8.9% penetrance. Clubfoot was unilateral in 16 of the 20 affected Pitx1(+/-) mice, with the right and left limbs equally affected, in contrast to right-sided predominant hindlimb abnormalities previously noted with complete loss of Pitx1. Peroneal artery hypoplasia occurred in the clubfoot limb and corresponded spatially with small lateral muscle compartments. Tibial and fibular bone volumes were also reduced. Skeletal muscle gene expression was significantly reduced in Pitx1(-/-) E12.5 hindlimb buds compared with the wild-type, suggesting that muscle hypoplasia was due to abnormal early muscle development and not disuse atrophy. Our morphological data suggest that PITX1 haploinsufficiency may cause a developmental field defect preferentially affecting the lateral lower leg, a theory that accounts for similar findings in human clubfoot.

Adaptation of tibial structure and strength to axial compression depends on loading history in both C57BL/6 and BALB/c mice.

Tibial compression can increase murine bone mass. However, loading protocols and mouse strains differ between studies, which may contribute to conflicting results. We hypothesized that bone accrual is influenced more by loading history than by mouse strain or animal handling. The right tibiae of 4-month-old C57BL/6 and BALB/c mice were subjected to axial compression (10 N, 3 days/week, 6 weeks). Left tibiae served as contralateral controls to calculate relative changes: (loaded - control)/control. The WashU protocol applied 60 cycles/day, at 2 Hz, with a 10-s rest-insertion between cycles; the Cornell/HSS protocol applied 1,200 cycles/day, at 6.7 Hz, with a 0.1-s rest-insertion. Because sham loading, sedation, and transportation did not affect tibial morphology, unhandled mice served as age-matched controls (AC). Both loading protocols were anabolic for cortical bone, but Cornell/HSS loading elicited a more rapid response that was greater than WashU loading by 13 %. By 6 weeks, cortical bone volume of each loading group was greater than of AC (average + 16 %) and not different from each other. Ultimate displacement and energy to fracture were greater in tibiae loaded by either protocol, and ultimate force was greater with Cornell/HSS loading. At 6 weeks, independent of mouse strain, the WashU protocol produced minimal trabecular bone and the trabecular bone volume fraction of Cornell/HSS tibiae was greater than that of AC by 65 % and that of WashU by 44 %. We concluded that tibial adaptation to loading was more influenced by waveform than mouse strain or animal handling and therefore may have targeted similar osteogenic mechanisms in C57BL/6 and BALB/c mice.

T cells heal bone fractures with help from the gut microbiome.

Immune cells play an important functional role in bone fracture healing. Fracture repair is a well-choreographed process that takes approximately 21 days in healthy mice. While the process is complex, conceptually it can be divided into four overlapping stages: inflammation, cartilaginous callus formation, bony callus formation, and remodeling. T cells play a key role in both the cartilaginous and bony callus phases by producing IL-17A. In this issue of the JCI, Dar et al. showed that T cells were recruited from the gut, where the gut microbiota determined the pool of T cells that expressed IL-17A. Treatment with antibiotics and dysbiosis reduced the expansion of IL-17-expressing CD4+ T cells (Th17) and impaired callus formation. These findings demonstrate crosstalk among the gut microbiota, the adaptive immune system, and bone that has clinical implications for fracture healing.

Multi-Scale Cortical Bone Traits Vary in Two Mouse Models of Genetic Diversity.

Understanding the genetic basis of cortical bone traits can allow for the discovery of novel genes or biological pathways regulating bone health. Mice are the most widely used mammalian model for skeletal biology and allow for the quantification of traits that can't easily be evaluated in humans, such as osteocyte lacunar morphology. The goal of our study was to investigate the effect of genetic diversity on multi-scale cortical bone traits of three long bones in skeletally-mature mice. We measured bone morphology, mechanical properties, material properties, lacunar morphology, and mineral composition of mouse bones from two populations of genetic diversity. Additionally, we compared how intra-bone relationships varied in the two populations. Our first population of genetic diversity included 72 females and 72 males from the eight Inbred Founder strains used to create the Diversity Outbred (DO) population. These eight strains together span almost 90% of the genetic diversity found in mice (Mus musculus). Our second population of genetic diversity included 25 genetically unique, outbred females and 25 males from the DO population. We show that multi-scale cortical bone traits vary significantly with genetic background; heritability values range from 21% to 99% indicating genetic control of bone traits across length scales. We show for the first time that lacunar shape and number are highly heritable. Comparing the two populations of genetic diversity, we show each DO mouse does not resemble a single Inbred Founder but instead the outbred mice display hybrid phenotypes with the elimination of extreme values. Additionally, intra-bone relationships (e.g., ultimate force vs. cortical area) were mainly conserved in our two populations. Overall, this work supports future use of these genetically diverse populations to discover novel genes contributing to cortical bone traits, especially at the lacunar length scale.

Biomechanical testing of fracture fixation constructs: variability, validity, and clinical applicability.

Biomechanical testing of fracture fixation implants is crucial in preclinical evaluation and in comparing new devices with standard devices. Many variables must be considered when planning and implementing a biomechanical in vitro experiment. The type of test selected (eg, load-to-failure, stiffness, cyclic fatigue) depends on the research question being asked. For example, cyclic fatigue testing attempts to replicate clinical situations; thus, the load magnitudes and directions and the number of cycles should be decided accordingly. Most important, each bone and region of bone experiences specific in vivo forces based on muscular and other forces. Debate persists regarding whether cadaver or synthetic bone is optimal. The use of either material in biomechanical testing should be carefully considered and justified in the context of the study hypothesis. Appropriate study design is the main factor that affects the clinical applicability of the findings and the accuracy of the conclusions.

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.

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 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.

Dmp1 Lineage Cells Contribute Significantly to Periosteal Lamellar Bone Formation Induced by Mechanical Loading But Are Depleted from the Bone Surface During Rapid Bone Formation.

Previous work has shown that osteoprogenitor cells (Prx1+) and pre-osteoblasts (Osx+) contribute to mechanical loading-induced bone formation. However, the role of mature Dmp1-expressing osteoblasts has not been reported. In this study we assessed the contribution of osteoblast lineage cells to bone formation at an early time point following mechanical loading (day 8 from onset of loading). We labeled Osx-expressing and Dmp1-expressing cells in inducible Osx and Dmp1 reporter mice (iOsx-Ai9, iDmp1-Ai9), respectively, 3 weeks before loading. Mice were then loaded daily for 5 days (days 1-5) and were dosed with 5-ethynyl-2'-deoxyuridine (EdU) in their drinking water until euthanasia on day 8. Mice were loaded to lamellar and woven bone inducing stimulation (-7 N/1400 µÎµ, -10 N/2000 µÎµ) to assess differences in these processes. We found varied responses in males and females to the loading stimuli, inducing modest lamellar (females, -7 N), moderate lamellar (males, -10 N), and robust woven (females, -10 N) bone. Overall, we found that preexisting (ie, lineage positive) Osx-expressing and Dmp1-expressing cells contribute largely to the bone formation response, especially during modest bone formation, while our results stuggest that other (non-lineage-positive) cells support the sustained bone formation response during rapid bone formation. With moderate or robust levels of bone formation, a decrease in preexisting Osx-expressing and Dmp1-expressing cells at the bone surface occurred, with a near depletion of Dmp1-expressing cells from the surface in female mice loaded to -10 N (from 52% to 11%). These cells appeared to be replaced by lineage-negative cells from the periosteum. We also found a dose response in proliferation, with 17% to 18% of bone surface cells arising via proliferation in modest lamellar, 38% to 53% in moderate lamellar, and 59% to 81% in robust woven bone formation. In summary, our results show predominant contributions by preexisting Osx and Dmp1 lineage cells to loading-induced lamellar bone formation, whereas recruitment of earlier osteoprogenitors and increased cell proliferation support robust woven bone formation. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Cortical bone relationships are maintained regardless of sex and diet in a large population of LGXSM advanced intercross mice.

Introduction: Knowledge of bone structure-function relationships in mice has been based on relatively small sample sets that limit generalizability. We sought to investigate structure-function relationships of long bones from a large population of genetically diverse mice. Therefore, we analyzed previously published data from the femur and radius of male and female mice from the F34 generation of the Large-by-Small advanced intercross line (LGXSM AI), which have over a two-fold continuous spread of bone and body sizes (Silva et al. 2019 JBMR). Methods: Morphological traits, mechanical properties, and estimated material properties were collected from the femur and radius from 1113 LGXSM AI adult mice (avg. age 25 wks). Males and females fed a low-fat or high-fat diet were evaluated to increase population variation. The data were analyzed using principal component analysis (PCA), Pearson's correlation, and multivariate linear regression. Results: Using PCA groupings and hierarchical clustering, we identified a reduced set of traits that span the population variation and are relatively independent of each other. These include three morphometry parameters (cortical area, medullary area, and length), two mechanical properties (ultimate force and post-yield displacement), and one material property (ultimate stress). When comparing traits of the femur to the radius, morphological traits are moderately well correlated (r2: 0.18-0.44) and independent of sex and diet. However, mechanical and material properties are weakly correlated or uncorrelated between the long bones. Ultimate force can be predicted from morphology with moderate accuracy for both long bones independent of variations due to genetics, sex, or diet; however, predictions miss up to 50 % of the variation in the population. Estimated material properties in the femur are moderately to strongly correlated with bone size parameters, while these correlations are very weak in the radius. Discussion: Our results indicate that variation in cortical bone phenotype in the F34 LGXSM AI mouse population can be adequately described by a reduced set of bone traits. These traits include cortical area, medullary area, bone length, ultimate force, post-yield displacement, and ultimate stress. The weak correlation of mechanical and material properties between the femur and radius indicates that the results from routine three-point bending tests of one long bone (e.g., femur) may not be generalizable to another long bone (e.g., radius). Additionally, these properties could not be fully predicted from bone morphology alone, confirming the importance of mechanical testing. Finally, material properties of the femur estimated based on beam theory equations showed a strong dependence on geometry that was not seen in the radius, suggesting that differences in femur size within a study may confound interpretation of estimated material properties.