Fracture Callus Cartilage Research Articles

Bifidobacterium adolescentis supplementation attenuates fracture-induced systemic sequelae

The gut microbiota is an important contributor to both health and disease. While previous studies have reported on the beneficial influences of the gut microbiota and probiotic supplementation on bone health, their role in recovery from skeletal injury and resultant systemic sequelae remains unexplored. This study aimed to determine the extent to which probiotics could modulate bone repair by dampening fracture-induced systemic inflammation. Our findings demonstrate that femur fracture induced an increase in gut permeability lasting up to 7 days after trauma before returning to basal levels. Strikingly, dietary supplementation with Bifidobacterium adolescentis augmented the tightening of the intestinal barrier, dampened the systemic inflammatory response to fracture, accelerated fracture callus cartilage remodeling, and elicited enhanced protection of the intact skeleton following fracture. Together, these data outline a mechanism whereby dietary supplementation with beneficial bacteria can be therapeutically targeted to prevent the systemic pathologies induced by femur fracture.

Deletion of FOXO1 in chondrocytes rescues the effect of diabetes on mechanical strength in fracture healing.

Diabetes increases the risk of fracture, impairs fracture healing and causes rapid loss of the fracture callus cartilage, which was linked to increased FOXO1 expression in chondrocytes. We recently demonstrated that deletion of FOXO1 in chondrocytes blocked the premature removal of cartilage associated with endochondral bone formation during fracture healing. However, the ultimate impact of this deletion on mechanical strength was not investigated and remains unknown. Closed fractures were induced in Col2α1Cre+.FOXO1L/L mice with lineage specific deletion of FOXO1 in chondrocytes compared to littermate controls. Type 1 diabetes was induced by multiple low dose streptozotocin treatment. Thirty-five days after fracture micro CT analysis showed that diabetes significantly reduced callus volume and bone volume (P < 0.05), both which were reversed by FOXO1 deletion in chondrocytes. Diabetes significantly reduced mechanical strength measured by maximum torque, stiffness, modulus of rigidity and toughness and FOXO1 deletion in diabetic mice rescued each parameter (P < 0.05). Diabetes also reduced both bone volume and mechanical strength in non-fractured femurs. However, FOXO1 deletion did not affect bone volume or strength in non-fractured bone. These results point to the important effect that diabetes has on chondrocytes and show for the first time that the premature removal of cartilage induced by FOXO1 in chondrocytes has a significant impact on the mechanical strength of the healing bone.

Changes in ephrin gene expression during bone healing identify a restricted repertoire of ephrinsmediating fracture repair.

To identify the repertoire of ephrin genes that might regulate endochondral bone fracture repair, we examined changes in ephrin ligand and receptor (Eph) gene expression in fracture callus tissues during bone fracture healing. Ephrin and Eph proteins were then localized in the fracture callus tissues present when changes in gene expression were observed. Ephrin gene expression was widespread in fracture tissues, but the repertoire of ephrin genes with significant changes in expression that might suggest a regulatory role in fracture callus development was restricted to the ephrin A family members Epha4, Epha5 and the ephrin B family member Efnb1. After 3 weeks of healing, Epha4 fracture expression was downregulated from 1.3- to 0.8-fold and Epha5 fracture expression was upregulated from 1.2- to 1.5-fold of intact contralateral femur expression, respectively. Efnb1 expression was downregulated from 1.5- to 1.2-fold after 2 weeks post-fracture. These ephrin proteins were localized to fracture callus prehypertrophic chondrocytes and osteoblasts, as well as to the periosteum and fibrous tissues. The observed positive correlation between mRNA levels of EfnB1 with Col10 and Epha5 with Bglap, together with colocalized expression with their respective proteins, suggest that EfnB1 is a positive mediator of prehypertrophic chondrocyte development and that Epha5 contributes to osteoblast-mediated mineralization of fracture callus. In contrast, mRNA levels of Epha4 and Efnb1 correlated negatively with Bglap, thus suggesting a negative role for these two ephrin family members in mature osteoblast functions. Given the number of family members and widespread expression of the ephrins, a characterization of changes in ephrin gene expression provides a basis for identifying ephrin family members that might regulate the molecular pathways of bone fracture repair. This approach suggests that a highly restricted repertoire of ephrins, EfnB1 and EphA5, are the major mediators of fracture callus cartilage hypertrophy and ossification, respectively, and proposes candidates for additional functional study and eventual therapeutic application.

Sclerostin monoclonal antibody enhanced bone fracture healing in an open osteotomy model in rats.

Sclerostin is a negative regulator of bone formation. Sclerostin monoclonal antibody (Scl-Ab) treatment promoted bone healing in various animal models. To further evaluate the healing efficiency of Scl-Ab in osteotomy healing, we investigated the time course effects of systemic administration of Scl-Ab on fracture repair in rat femoral osteotomy model. A total of 120 six-month-old male SD rats were subjected to transverse osteotomy at the right femur mid-shaft. Rats were treated with vehicle or Scl-Ab treatment for 3, 6, or 9 weeks. Fracture healing was evaluated by radiography, micro-CT, micro-CT based angiography, 4-point bending mechanical test and histological assessment. Scl-Ab treatment resulted in significantly higher total mineralized callus volume fraction, BMD and enhanced neovascularization. Histologically, Scl-Ab treatment resulted in a significant reduction in fracture callus cartilage at week 6 and increase in bone volume at week 9, associated with a greater proportion of newly formed bone area at week 6 and 9 by fluorescence microscopy. Mechanical testing showed significantly higher ultimate load in Scl-Ab treatment group at week 6 and 9. This study has demonstrated that Scl-Ab treatment enhanced bone healing in a rat femoral osteotomy model, as reflected in increased bone formation, bone mass and bone strength.

Duffy antigen receptor for chemokines regulates post-fracture inflammation.

There is now considerable experimental data to suggest that inflammatory cells collaborate in the healing of skeletal fractures. In terms of mechanisms that contribute to the recruitment of inflammatory cells to the fracture site, chemokines and their receptors have received considerable attention. Our previous findings have shown that Duffy antigen receptor for chemokines (Darc), the non-classical chemokine receptor that does not signal, but rather acts as a scavenger of chemokines that regulate cell migration, is a negative regulator of peak bone density in mice. Furthermore, because Darc is expressed by inflammatory and endothelial cells, we hypothesized that disruption of Darc action will affect post-fracture inflammation and consequently will affect fracture healing. To test this hypothesis, we evaluated fracture healing in mice with targeted disruption of Darc and corresponding wild type (WT) control mice. We found that fracture callus cartilage formation was significantly greater (33%) at 7 days post-surgery in Darc-KO compared to WT mice. The increased cartilage was associated with greater Collagen (Col) II expression at 3 days post-fracture and Col-X at 7 days post-fracture compared to WT mice, suggesting that Darc deficiency led to early fracture cartilage formation and differentiation. We then compared the expression of cytokine and chemokine genes known to be induced during inflammation. Interleukin (Il)-1β, Il-6, and monocyte chemotactic protein 1 were all down regulated in the fractures derived from Darc-KO mice at one day post-fracture, consistent with an altered inflammatory response. Furthermore, the number of macrophages was significantly reduced around the fractures in Darc-KO compared to WT mice. Based on these data, we concluded that Darc plays a role in modulating the early inflammatory response to bone fracture and subsequent cartilage formation. However, the early cartilage formation was not translated with an early bone formation at the fracture site in Darc-KO compared to WT mice.

Urokinase plasminogen activator gene deficiency inhibits fracture cartilage remodeling

Urokinase plasminogen activator (uPA) regulates a proteolytic cascade of extracellular matrix degradation that functions in tissue development and tissue repair. The development and remodeling of the skeletal extracellular matrix during wound healing suggests that uPA might regulate bone development and repair. To determine whether uPA functions regulate bone development and repair, we examined the basal skeletal phenotype and endochondral bone fracture repair in uPA-deficient mice. The skeletal phenotype of uPA knockout mice was compared with that of control mice under basal conditions by dual-energy X-ray absorptiometry and micro-CT analysis, and during femur fracture repair by micro-CT and histological examination of the fracture callus. No effects of uPA gene deficiency were observed in the basal skeletal phenotype of the whole body or the femur. However, uPA gene deficiency resulted in increased fracture callus cartilage abundance during femur fracture repair at 14days healing. The increase in cartilage corresponded to reduced tartrate-resistant acid phosphatase (TRAP) staining for osteoclasts in the uPA knockout fracture callus at this time, consistent with impaired osteoclast-mediated remodeling of the fracture cartilage. CD31 staining was reduced in the knockout fracture tissues at this time, suggesting that angiogenesis was also reduced. Osteoclasts also colocalized with CD31 expression in the endothelial cells of the fracture tissues during callus remodeling. These results indicate that uPA promotes remodeling of the fracture cartilage by osteoclasts that are associated with angiogenesis and suggest that uPA promotes angiogenesis and remodeling of the fracture cartilage at this time of bone fracture repair.

Immunolocalization of Myostatin (GDF-8) Following Musculoskeletal Injury and the Effects of Exogenous Myostatin on Muscle and Bone Healing

The time course and cellular localization of myostatin expression following musculoskeletal injury are not well understood; therefore, the authors evaluated the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. They then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results showed that a "pool" of intense myostatin staining was observed among injured skeletal muscle fibers 12-24 hr postsurgery and that myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 µg/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also decreased fracture callus total bone volume by 30.6% and 38.8% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury.

Fracture Healing in Mice Deficient in Plasminogen Activator Inhibitor-1

To evaluate the role of plasminogen activator inhibitor (PAI)-1, a key negative regulator of the plasmin system of extracellular matrix proteases in developmental bone growth and fracture repair, the bone phenotype of male adult PAI-1-deficient mice was determined and femoral fracture healing was compared with that of age- and sex-matched wild-type C57BL/6J control mice. Regarding bone phenotype, the length and size (but not cortical thickness) of the femur of male PAI-1-deficient mice were smaller than those of wild-type controls. Although the total bone mineral content of PAI-1-deficient mice was not significantly different from that of wild-type mice, the total bone area in PAI-1-deficient mice was smaller, leading to an increase in total bone mineral density. With respect to fracture healing, PAI-1-deficient mice developed fracture calluses that were larger and more mineralized than those of wild-type mice but only at 14 days postfracture. These changes were even greater given the smaller size of the normal femur in PAI-1-deficient mice. Surprisingly, the larger fracture callus remodeled rapidly to normal size and mineral content by 21 days postfracture. Examination of fracture histology revealed that these changes were associated with a dramatic increase followed by a rapid remodeling of the fracture callus cartilage. The remodeling of fracture callus cartilage in PAI-1-deficient mice also displayed an abnormal pattern. These findings demonstrate for the first time that PAI-1 (and potentially the plasminogen extracellular matrix protease system) is an important regulator of bone size during developmental growth and plays a regulatory role in the determination of fracture callus size, cartilage formation, and resorption during bone fracture repair.

794. Retroviral-Based Gene Therapy with Cyclo-Oxygenase-2 Promotes Bony Union and Accelerates Fracture Healing in the Rat

Fracture repair of endochondral bones involves an ordered series of processes, including the proliferation of fibrous tissues, their replacement by cartilage, and the conversion of cartilage to bony tissue that ultimately bridges the fracture. While recent gene therapy studies with osteogenic genes, e.g., BMPs, yielded encouraging results in that they augmented bone production at the fracture site, none of the published strategies to date have accelerated bony union of the fracture gap, which is the hallmark of successful fracture repair. In this study, we evaluated the efficacy of cyclo-oxygenase-2 (Cox-2) gene therapy in promoting bony union in a rat femur fracture model. The Cox-2 gene was chosen because Cox-2 promotes inflammation, angiogenesis, bone formation and bone resorption; all of these processes are essential for fracture repair. However, previous gene therapy with Cox-2 has been limited by the short half-life of Cox-2 mRNA, a result of the destabilizing ARE domains of the 3|[prime]| untranslated region (3|[prime]|UTR). Thus, we modified the human Cox-2 transgene by deleting its 3|[prime]|UTR and by including an optimized Kozak sequence to increase the stability of Cox-2 mRNA and to enhance Cox-2 protein translation, respectively. A murine leukemia virus (MLV)-based retroviral vector was used to target the expression of the Cox-2 transgene or a |[beta]|-galactosidase control transgene to the cells of the periosteum induced to proliferate by the injury. The retroviral vector was injected directly into the periosteum at the fracture site in a single percutaneous injection at one day post-fracture. Healing was evaluated by X-ray analysis and bone histomorphometry. Real-time RT-PCR analysis of human Cox-2 gene expression in Cox-2 and control fracture tissues at 4, 7, 14 and 21 days healing (N=4 each) established that human Cox-2 transgene expression significantly increased total Cox-2 gene expression at each time point, and greater than 10-fold at 21 days (p<0.03). Endogenous rat Cox-2 gene expression was unaffected by the treatment. X-ray and histology analysis revealed that, unlike BMP therapy, Cox-2 gene therapy produced a symmetric callus around the entire circumference of the fracture without supra-periosteal bone formation. More importantly, 7 of 8 MLV-Cox-2 treated fractures yielded bony union of the fracture callus at 21 days, while only 1 of 6 MLV-|[beta]|-galactosidase treated fractures showed evidence of bony union. This healing occurred well before the 4 to 5 weeks normally required for bony union in this fracture model. Histomorphometric measurements demonstrated that bony union coincided with a reduction in the cartilage area from 3.7% to 0.6% (p<0.005) within the fracture site at 21 days healing, indicating that Cox-2 gene therapy accelerated the replacement of the fracture callus cartilage with bone to generate earlier bony union of the fracture. Sinusoid-like structures in Cox-2 treated fractures suggested that Cox-2 therapy promoted angiogenesis. In summary, we have demonstrated for the first time that local expression of a Cox-2 transgene from a retroviral vector accelerated bony union in a normally healing fracture model.

Early histologic and ultrastructural changes in microvessels of periosteal callus.

To document early histological and ultrastructural changes in periosteal fracture callus blood vessels. Rabbit control and fractured ribs, after healing for three, six, and twelve hours and daily for seven days, were evaluated by light and electron microscopy. Control periosteal microvessels were formed mainly by endothelial cells and occasionally by pericytes. Only these cells displayed basal lamina within the periosteum. Three to twelve hours postfracture, periosteal microvessels were little changed. By two days postfracture, dramatic increases in size and population of microvessel cells resulted in a smaller lumen and thicker wall. Microvessel cells, while retaining their basal lamina, had transformed to mesenchymal cells. Transformed pericytes, as evidenced by their basal lamina, had extravasated. Three to four days postfracture, additional transformed pericytes had extravasated. Within the distal periosteal callus, a close spatial relationship among transformed microvessels, extravascular mesenchymal cells (some with basal lamina), and osteoblasts was present. Four to five days postfracture, within the proximal periosteal callus, a close spatial relationship among transformed microvessels (rapidly disappearing because of continued extravasation), extravascular mesenchymal cells (some with basal lamina), and chondroblasts (some with basal lamina) was present. New evidence showed that after fracture, periosteal microvessel endothelial cells and pericytes increased in population and transformed to mesenchymal cells. These changes, their subsequent extravasation as mesenchymal cells, and their development into chondroblasts were verified by basal lamina evidence. New evidence also suggested that continued extravasation of transformed microvessel cells rendered the fracture callus cartilage avascular.

Purification of alkaline phosphatase from extracellular vesicles of fracture callus cartilage.

Extracellular matrix vesicles from fracture callus cartilage were isolated by differential centrifugation and resolved by equilibrium centrifugation on a discontinuous sucrose gradient into two bands. The phosphohydrolytic activity towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was distributed similarly after differential and equilibrium centrifugation suggesting the association of this activity with the matrix vesicles. The two bands isolated by equilibrium centrifugation of the partially purified vesicular preparation demonstrated high levels of alkaline phosphatase activity. Observed with an electron microscope, the 1.07–1.14 g/cm3 band from the gradient was enriched in electron luscent matrix vesicles while the 1.27 g/cm3 band contained electron dense matrix vesicles. Enzymatic analysis of the 1.27 g/cm3 band indicated a slight contamination due to the presence of mitochondria and lysosomes while the 1.07–1.14 g/cm3 band gave no enzymatic indication of subcellular contamination. A phosphohydrolytic enzyme active towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was purified from the 1.07–1.14 g/cm3 fraction by DEAE-cellulose column chromatography. Electron micrographs of callus cartilage sections demonstrated densification of the plasma membrane and matrix vesicles following substrate incubation withβ-glycerophosphate or tetrasodium pyrophosphate. The histochemical and biochemical data indicate that a phosphatase, with multiple substrate specificity, is a component of fracture callus cartilage matrix vesicles.

Studies on the Mechanism of Callus Cartilage Differentiation and Calcification During Fracture Healing

The morphologic and biochemical events during fracture callus cartilage differentiation and calcification are presented. 1. Histologic studies have demonstrated that unimmobilized fractures heal through endochondral ossification. 2. Biochemical studies have demonstrated an increase in the activities of alkaline phosphatase, enzymes involved with aerobic glucose metabolism, and lysosomal enzymes and a decrease in activities of enzymes involved with glycogen synthesis and anaerobic glycolysis. Hexosamines and hydroxyproline show a net decrease with cartilage differentiation. 3. Electron microscopic studies have demonstrated the intracellular origin and aggregation of collagen molecules, the cellular origin of matrix vesicles, and the early sites of calcification in the fracture callus.

Scanning Electron Microscopy of Chondrocytes from Fracture Callus Cartilage

There is little information available in the literature on scanning electron microscopy of cartilage tissue. Most of the available information deals with articular cartilage (1,2). More specifically, there are no studies reported on the scanning electron microscopy of differentiating callus cartilage. This preliminary report is part of our studies on the differentiation and eventual calcification of rabbit callus cartilage.Closed fractures were produced in the tibias of white rabbits by acute angulation. Seven days after fracture, the callus was dissected into homogenous cartilage sections corresponding to the fibrous, hypertrophic and calcified cartilage types of the epiphyseal plate (3). The cartilage specimens were dehydrated by ascending acetone series or freeze-dried. Some acetone dried specimens were freeze-dried while others were critical point dried. All specimens were mounted on Cambridge specimen stubs and coated with carbon and gold.

The biochemical activity of fracture callus in relation to bone production.

Quantitative microchemical study of the tissues comprising fracture callus has been undertaken to correlate the biochemical activity of the bone repair process with its previously established morphological features. Areas of proliferating fibrous tissue, hypertrophic cartilage, new bone and undifferentiated granulation tissue were analyzed for their content of carbohydrate metabolizing and phosphatase enzymes. Fracture callus cartilage is biochemically similar to epiphyseal cartilage. Carbohydrate metabolism provides structural intermediates and energy for bone repair. Inorganic pyrophosphatase removes the inorganic pyrophosphate which accumulates from structural synthesis and prevents its inhibition of new bone calcification. The individual parts of the callus have identical biochemical function regardless of the age or healing time of the fracture callus.

Morphological and biochemical studies during differentiation and calcification of fracture callus cartilage.

Differentiation and calcification of cartilage of a fracture callus morphologically, ultrastructurally, and histochemically resembles cartilage of growing epiphyseal plate. The fracture callus includes the various cartilage cell types found in the epiphyseal plate. Proliferating and hypertrophic cartilage had higher activities of cytochrome oxidase, alkaline phosphatase and glutamate aspartate transaminase than fibrocartilage. Enzymes controlling glycogen synthesis and glycolysis had higher levels of activity in fibrocartilage than in hypertrophic cartilage. Lysosomal enzymes, catalase, 6-phospho-gluconic acid and glucose 6-phosphate dehydrogenase were uniformly distributed. Alkaline phosphatase was associated with extracellular vesicles found in hypertrophic cartilage. EM dense granules were found in mitochondria in hypertrophic cartilage. There was an increase of total lipids in hypertropic and calcified cartilage as compared to resting cartilage.

Fracture callus cartilage differentiation in vitro.

An in vitro model has been devised to study the differentiation and calcification of fibrocartilage from fracture calluses. Morphological and biochemical studies substantiating cartilage metaplasia in vitro are presented. A role that such a model may play in the study of cartilage calcification is suggested.