Postnatal Osterix but not DMP1 lineage cells significantly contribute to intramembranous ossification in three preclinical models of bone injury.

Murine models of long-bone fracture, stress fracture, and cortical defect are used to discern the cellular and molecular mediators of intramembranous and endochondral bone healing. Previous work has shown that Osterix (Osx+) and Dentin Matrix Protein-1 (DMP1+) lineage cells and their progeny contribute to injury-induced woven bone formation during femoral fracture, ulnar stress fracture, and tibial cortical defect repair. However, the contribution of pre-existing versus newly-derived Osx+ and DMP1+ lineage cells in these murine models of bone injury is unclear. We addressed this knowledge gap by using male and female 12-week-old, tamoxifen-inducible Osx Cre_ERT2 and DMP1 Cre_ERT2 mice harboring the Ai9 TdTomato reporter allele. To trace pre-existing Osx+ and DMP1+ lineage cells, tamoxifen (TMX: 100 mg/kg gavage) was given in a pulse manner (three doses, 4 weeks before injury), while to label pre-existing and newly-derived lineage Osx+ and DMP1+ cells, TMX was first given 2 weeks before injury and continuously (twice weekly) throughout healing. TdTomato positive (TdT+) cell area and cell fraction were quantified from frozen histological sections of injured and uninjured contralateral samples at times corresponding with active woven bone formation in each model. We found that in uninjured cortical bone tissue, Osx Cre_ERT2 was more efficient than DMP1 Cre_ERT2 at labeling the periosteal and endosteal surfaces, as well as intracortical osteocytes. Pulse-labeling revealed that pre-existing Osx+ lineage and their progeny, but not pre-existing DMP1+ lineage cells and their progeny, significantly contributed to woven bone formation in all three injury models. In particular, these pre-existing Osx+ lineage cells mainly lined new woven bone surfaces and became embedded as osteocytes. In contrast, with continuous dosing, both Osx+ and DMP1+ lineage cells and their progeny contributed to intramembranous woven bone formation, with higher TdT+ tissue area and cell fraction in Osx+ lineage versus DMP1+ lineage calluses (femoral fracture and ulnar stress fracture). Similarly, Osx+ and DMP1+ lineage cells and their progeny significantly contributed to endochondral callus regions with continuous dosing only, with higher TdT+ chondrocyte fraction in Osx+ versus DMP1+ cell lineages. In summary, pre-existing Osx+ but not DMP1+ lineage cells and their progeny make up a significant amount of woven bone cells (particularly osteocytes) across three preclinical models of bone injury. Therefore, Osx+ cell lineage modulation may prove to be an effective therapy to enhance bone regeneration.

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model.

Nonunion is defined as the permanent failure of a fractured bone to heal, often necessitating surgical intervention. Atrophic nonunions are a subtype that are particularly difficult to treat. Animal models of atrophic nonunion are available; however, these require surgical or radiation-induced trauma to disrupt periosteal healing. These methods are invasive and not representative of many clinical nonunions where osseous regeneration has been arrested by a "failure of biology". We hypothesized that arresting osteoblast cell proliferation after fracture would lead to atrophic nonunion in mice. Using mice that express a thymidine kinase (tk) "suicide gene" driven by the 3.6Col1a1 promoter (Col1-tk), proliferating osteoblast lineage cells can be ablated upon exposure to the nucleoside analog ganciclovir (GCV). Wild-type (WT; control) and Col1-tk littermates were subjected to a full femur fracture and intramedullary fixation at 12 weeks age. We confirmed abundant tk+ cells in fracture callus of Col-tk mice dosed with water or GCV, specifically many osteoblasts, osteocytes, and chondrocytes at the cartilage-bone interface. Histologically, we observed altered callus composition in Col1-tk mice at 2 and 3 weeks postfracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and micro-computed tomography (µCT) scans, had delayed bone bridging and reduced callus size. After euthanasia, ex vivo µCT and histology showed failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing showed a failure to recover torsional strength in Col1-tk mice, in contrast to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for at least 2 weeks after fracture blunts the formation and remodeling of a mineralized callus leading to a functional nonunion. We propose this as a new murine model of atrophic nonunion. © 2021 American Society for Bone and Mineral Research (ASBMR).

Flexor Tendon Injury and Repair. The Influence of Synovial Environment on the Early Healing Response in a Canine Model.

BACKGROUND: Environmental conditions strongly influence the healing capacity of connective tissues. Well-vascularized extrasynovial tendons typically undergo a robust wound-healing process following transection and repair. In contrast, avascular intrasynovial tendons do not mount an effective repair response. The current study tests the hypothesis that flexor tendons, as a function of their synovial environment, exhibit unique inflammatory, angiogenic, and metabolic responses to injury and repair. METHODS: Flexor tendons present a distinct opportunity to test the study hypothesis, as they have proximal regions that are extrasynovial and distal regions that are intrasynovial. In an internally controlled study design, the second and fifth forepaw flexor tendons were transected and repaired in either the extrasynovial or the intrasynovial anatomical region. Histological, gene expression, and proteomics analyses were performed at 3 and 7 days to define the early biological events that drive synovial environment-dependent healing responses. RESULTS: Uninjured intrasynovial tendons were avascular, contained high levels of proteoglycans, and expressed inflammatory factors, complement proteins, and glycolytic enzymes. In contrast, extrasynovial tendons were well vascularized, contained low levels of proteoglycans, and were enriched in inflammation inhibitors and oxidative phosphorylation enzymes. The response to injury and repair was markedly different between the 2 tendon regions. Extrasynovial tendons displayed a robust and rapid neovascularization response, increased expression levels of complement proteins, and an acute shift in metabolism to glycolysis, whereas intrasynovial tendons showed minimal vascularity and muted inflammatory and metabolic responses. CONCLUSIONS: The regional molecular profiles of intact and healing flexor tendons revealed extensive early differences in innate immune response, metabolism, vascularization, and expression of extracellular matrix as a function of the synovial environment. These differences reveal mechanisms through which extrasynovial tendons heal more effectively than do intrasynovial tendons. CLINICAL RELEVANCE: To improve outcomes after operative repair, future treatment strategies should promote features of extrasynovial healing, such as enhanced vascularization and modulation of the complement system and/or glucose metabolism.

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.

Stem cell-derived extracellular vesicles attenuate the early inflammatory response after tendon injury and repair.

Adipose-derived stem cells (ASCs) have the potential to enhance tendon repair via paracrine regulation of the inflammatory response to injury. Extracellular vesicles (EVs), which are secreted by ASCs, have shown promise in mediating this process. This study was designed to evaluate the effect of ASC EVs on early tendon healing using a mouse Achilles tendon injury and repair model. EVs were isolated from the conditioned medium of naïve and interferonγ-primed ASCs and applied to the repair site via a collagen sheet. Tendon healing was assessed in nuclear factor-κB (NF-κB)-luciferase reporter mice up to 7 days after suture repair. As anticipated, repair site NF-κB activity increased greater than twofold following tendon repair. Treatment with EVs from primed but not naïve ASCs effectively suppressed the response. Accordingly, the pro-inflammatory genes Il1b and Ifng were both dramatically increased in repaired tendons, while primed, but not naïve ASC EVs attenuated the response. Compared with control repairs, primed ASC EVs further reduced the rate of post-repair tendon gap formation and rupture and facilitated collagen formation at the injury site. Additional experiments demonstrated that EVs target macrophages and that primed ASC EVs were most effective in blocking macrophage NF-κB activity. Collectively, the findings of this study demonstrate that primed ASC EVs, similar to ASCs, attenuate the early tendon inflammatory response after injury via modulation of the macrophage inflammatory response. Statement of clinical significance: These findings introduce a new cell-free therapy, derived from stem cells, for tendon repair with the potential for improved therapeutic efficacy and safety. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:117-127, 2020.

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.

The effect of modified locking methods and suture materials on Zone II flexor tendon repair-An ex vivo study.

The failure rate of intrasynovial tendon repair is high due to substantial elongation at the repair site and to the development of adhesions between the tendon's surface and the surrounding digital sheath. To minimize these complications, we sought to reduce the incidence of gapping and to facilitate the initiation of early motion by improving the time zero structural properties of repair. The Winters-Gelberman 8-strand repair technique was modified by adding surface lock loops and by using Fiberwire suture material. Forty-eight canine flexor digitorum profundus tendons were transected and repaired with one of three 8-strand techniques (Pennington modified Kessler, half hitch loops, or surface locking Kessler) using either 3-0 Supramid or 4-0 Fiberwire suture. Biomechanical testing was performed to determine the physiologic and failure mode properties of the repairs. The surface locking Kessler technique improved repair maximum load, load necessary to create a 2 mm repair site gap, and yield force compared to the modified Kessler and half hitch loop techniques. Fiberwire suture improved maximum load, the load necessary to create a 2 mm repair site gap, stiffness, and yield force compared to Supramid suture. Failure occurred by both suture pull out and by suture breakage in the modified Kessler, Supramid suture repair group. Failure occurred consistently by suture breakage in the surface locking Kessler, Supramid suture repair group. These results reveal that a novel locking Kessler repair is significantly stronger than the current state-of-the art flexor tendon suture repair technique. The use of a surface locking Kessler technique with Fiberwire suture markedly improves the mechanical properties of intrasynovial tendon repair by reducing the risk of post-operative gapping and rupture.

The effect of adipose-derived stem cell sheets and CTGF on early flexor tendon healing in a canine model.

Intrasynovial tendon injuries are among the most challenging in orthopedics. Despite significant improvements in operative and rehabilitation methods, functional outcomes continue to be limited by adhesions, gap formation, and rupture. Adhesions result from excessive inflammation, whereas tendon gapping and rupture result from inflammation-induced matrix degradation and insufficient regeneration. Therefore, this study used a combined treatment approach to modulate inflammation with adipose-derived mesenchymal stromal cells (ASCs) while stimulating tendon regeneration with connective tissue growth factor (CTGF). ASCs were applied to the repair surface via cell sheets and CTGF was delivered to the repair center via porous sutures. The effect of the combined treatment was assessed fourteen days after repair in a canine flexor tendon injury model. CTGF, either alone or with ASCs, reduced inflammatory (IL1B and IL6) and matrix degrading (MMP3 and MMP13) gene expression, while increasing anti-inflammatory gene (IL4) expression and collagen synthesis compared to control repairs. The combined treatment was more effective than CTGF treatment alone, reducing the inflammatory IFNG and scar-associated COL3A1 gene expression and increasing CD146+ tendon stem/progenitor cells at the tendon surface and interior along the core suture tracks. Therefore, the combined approach is promising in promoting early flexor tendon healing and worthy of further investigation.

Effect of connective tissue growth factor delivered via porous sutures on the proliferative stage of intrasynovial tendon repair.

Recent growth factor, cell, and scaffold-based experimental interventions for intrasynovial flexor tendon repair have demonstrated therapeutic potential in rodent models. However, these approaches have not achieved consistent functional improvements in large animal trials due to deleterious inflammatory reactions to delivery materials and insufficient induction of targeted biological healing responses. In this study, we achieved porous suture-based sustained delivery of connective tissue growth factor (CTGF) into flexor tendons in a clinically relevant canine model. Repairs with CTGF-laden sutures were mechanically competent and did not show any evidence of adhesions or other negative inflammatory reactions based on histology, gene expression, or proteomics analyses at 14 days following repair. CTGF-laden sutures induced local cellular infiltration and a significant biological response immediately adjacent to the suture, including histological signs of angiogenesis and collagen deposition. There were no evident widespread biological effects throughout the tendon substance. There were significant differences in gene expression of the macrophage marker CD163 and anti-apoptotic factor BCL2L1; however, these differences were not corroborated by proteomics analysis. In summary, this study provided encouraging evidence of sustained delivery of biologically active CTGF from porous sutures without signs of a negative inflammatory reaction. With the development of a safe and effective method for generating a positive local biological response, future studies can explore additional methods for enhancing intrasynovial tendon repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2052-2063, 2018.