Natural Fracture Healing Research Articles

Advances in Dynamization of Plate Fixation to Promote Natural Bone Healing.

The controlled dynamization of fractures can promote natural fracture healing by callus formation, while overly rigid fixation can suppress healing. The advent of locked plating technology enabled new strategies for the controlled dynamization of fractures, such as far cortical locking (FCL) screws or active plates with elastically suspended screw holes. However, these strategies did not allow for the use of non-locking screws, which are typically used to reduce bone fragments to the plate. This study documents the first in vivo study on the healing of ovine tibia osteotomies stabilized with an advanced active plate (AAP). This AAP allowed plate application using any combination of locking and non-locking screws to support a wide range of plate application techniques. At week 9 post-surgery, tibiae were harvested and tested in torsion to failure to assess the healing strength. The five tibiae stabilized with an AAP regained 54% of their native strength and failed by spiral fracture through a screw hole, which did not involve the healed osteotomy. In comparison, tibiae stabilized with a standard locking plate recovered 17% of their strength and sustained failure through the osteotomy. These results further support the stimulatory effect of controlled motion on fracture healing. As such, the controlled dynamization of locked plating constructs may hold the potential to reduce healing complications and may shorten the time to return to function. Integrating controlled dynamization into fracture plates that support a standard fixation technique may facilitate the clinical adoption of dynamic plating.

Prx1-expressing cells contributing to fracture repair require primary cilia for complete healing in mice.

Bone is a dynamic organ that is continuously modified during development, load-induced adaptation, and fracture repair. Understanding the cellular and molecular mechanisms for natural fracture healing can lead to therapeutics that enhance the quality of newly formed tissue, advance the rate of healing, or replace the need for invasive surgical procedures. Prx1-expressing cells in the periosteum are thought to supply the majority of osteoblasts and chondrocytes in the fracture callus, but the exact mechanisms for this behavior are unknown. The primary cilium is a sensory organelle that is known to mediate several signaling pathways involved in fracture healing and required for Prx1-expressing cells to contribute to juvenile bone development and adult load-induced bone formation. We therefore investigated the role of Prx1-expressing cell primary cilia in fracture repair by developing a mouse model that enabled us to simultaneously track Prx1 lineage cell fate and disrupt Prx1-expressing cell primary cilia in vivo. The cilium KO mice exhibited abnormally large calluses with significantly decreased bone formation and persistent cartilage nodules. Analysis of mRNA expression in the early soft callus revealed downregulation of osteogenesis, Hh signaling, and Wnt signaling, and upregulation of chondrogenesis and angiogenesis. The mutant mice also exhibited decreased Osx and Periostin but increased αSMA and PECAM-1 protein expression in the hard callus. We further used a Gli1LacZ reporter and found that Hh signaling was significantly upregulated in the mutant callus at later stages of healing. Interestingly, altered protein expression and Hh signaling did not correlate with labeled Prx1-lineage cells, suggesting loss of cilia altered Hh signaling non-autonomously. Overall, cilium KO mice demonstrated severely delayed and incomplete fracture healing, and our findings suggest Prx1-expressing cell primary cilia are necessary to tune Hh signaling for proper fracture repair.

Developmentally Engineered Callus Organoid Bioassemblies Exhibit Predictive In Vivo Long Bone Healing.

Clinical translation of cell‐based products is hampered by their limited predictive in vivo performance. To overcome this hurdle, engineering strategies advocate to fabricate tissue products through processes that mimic development and regeneration, a strategy applicable for the healing of large bone defects, an unmet medical need. Natural fracture healing occurs through the formation of a cartilage intermediate, termed “soft callus,” which is transformed into bone following a process that recapitulates developmental events. The main contributors to the soft callus are cells derived from the periosteum, containing potent skeletal stem cells. Herein, cells derived from human periosteum are used for the scalable production of microspheroids that are differentiated into callus organoids. The organoids attain autonomy and exhibit the capacity to form ectopic bone microorgans in vivo. This potency is linked to specific gene signatures mimicking those found in developing and healing long bones. Furthermore, callus organoids spontaneously bioassemble in vitro into large engineered tissues able to heal murine critical‐sized long bone defects. The regenerated bone exhibits similar morphological properties to those of native tibia. These callus organoids can be viewed as a living “bio‐ink” allowing bottom‐up manufacturing of multimodular tissues with complex geometric features and inbuilt quality attributes.

Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair.

Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation, directed by local morphogen signals and mechanical cues. Here, we aimed to mimic development for regeneration of large bone defects. We hypothesized that engineered human mesenchymal condensations presenting transforming growth factor-β1 (TGF-β1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles promotes endochondral defect regeneration contingent on in vivo mechanical cues. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with BMP-2 + TGF-β1 fully restoring mechanical function. Delayed in vivo ambulatory loading significantly enhanced the bone formation rate in the dual morphogen group. In vitro, BMP-2 or BMP-2 + TGF-β1 initiated robust endochondral lineage commitment. In vivo, however, extensive cartilage formation was evident predominantly in the BMP-2 + TGF-β1 group, enhanced by mechanical loading. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.

Recapitulating bone development through engineered mesenchymal condensations and mechanical cues for tissue regeneration.

Large bone defects cannot form a callus and exhibit high complication rates even with the best treatment strategies available. Tissue engineering approaches often use scaffolds designed to match the properties of mature bone. However, natural fracture healing is most efficient when it recapitulates development, forming bone via a cartilage intermediate (endochondral ossification). Because mechanical forces are critical for proper endochondral bone development and fracture repair, we hypothesized that recapitulating developmental mechanical forces would be essential for large bone defect regeneration in rats. Here, we engineered mesenchymal condensations that mimic the cellular organization and lineage progression of the early limb bud in response to local transforming growth factor-β1 presentation from incorporated gelatin microspheres. We then controlled mechanical loading in vivo by dynamically tuning fixator compliance. Mechanical loading enhanced mesenchymal condensation-induced endochondral bone formation in vivo, restoring functional bone properties when load initiation was delayed to week 4 after defect formation. Live cell transplantation produced zonal human cartilage and primary spongiosa mimetic of the native growth plate, whereas condensation devitalization before transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose bone morphogenetic protein-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity. In vitro, mechanical loading promoted chondrogenesis and up-regulated pericellular matrix deposition and angiogenic gene expression. In vivo, mechanical loading regulated cartilage formation and neovascular invasion, dependent on load timing. This study establishes mechanical cues as key regulators of endochondral bone defect regeneration and provides a paradigm for recapitulating developmental programs for tissue engineering.

Augmentation of fracture healing by hydroxyapatite/collagen paste and bone morphogenetic protein-2 evaluated using a rat femur osteotomy model.

In fracture treatment, biological bone union generally depends on the bone's natural fracture healing capacity, even in surgically treated cases. Hydroxyapatite/collagen composite (HAp/Col) has high osteoconductivity and stimulates osteogenic progenitors. Furthermore, it has the potent capacity to adsorb bone morphogenetic proteins (BMPs). In this study, we prepared an injectable HAp/Col paste and evaluated its augmentation of bone union. Furthermore, the effect of HAp/Col paste combined with BMP-2 was also evaluated. We used a rat femur osteotomy model with a defect size of 1 mm. Male Wistar rats were assigned to one of the following four groups; a control group without any implant, a HAp/Col implant group, a group that received an absorbable collagen sponge (ACS) implant impregnated with BMP-2 (1 μg), and a group that received a HAp/Col implant impregnated with BMP-2 implant. Micro-CT analysis, three-point bending tests, and histological evaluation were performed. Bone union was achieved in two of eight cases in the HAp/Col group, five of eight cases in the ACS + BMP-2 group, and all cases in the HAp/Col + BMP-2 group at 8 weeks post-surgery. The control group did not achieve bone union. In addition, in the HAp/Col + BMP-2 group, the biomechanical strength of the fused femurs was comparable to that of the contralateral intact femur; the ratio of the mechanical load at the breaking point of the osteotomy side relative to that of the contralateral side was 1.00 ± 0.151 (SD). These results indicate that HAp/Col paste with or without BMP-2 augments bone union. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:129-137, 2018.

Bone fracture and the healing mechanisms. The effect of human parathyroid hormone on fracture healing

We reviewed the effect of human parathyroid hormone (hPTH) on fracture healing. In rodent fracture model, intermittent treatment of PTH accelerated the healing process as evidenced by earlier replacement of woven bone to lamellar bone, increased new cortical shell formation, and increased the ultimate load. In cynomolgus monkey fracture model, PTH accelerates the natural fracture healing process by shrinking callus size and increasing degree of mineralization of the fracture callus, thereby restoring intrinsic material properties.

Human parathyroid hormone (1–34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys

Several studies in rats have demonstrated that parathyroid hormone accelerates fracture healing by increasing callus formation or stimulating callus remodeling. However the effect of PTH on fracture healing has not been tested using large animals with Haversian remodeling system. Using cynomolgus monkey that has intracortical remodeling similar to humans, we examined whether intermittent treatment with human parathyroid hormone [hPTH(1–34)] accelerates the fracture healing process, especially callus remodeling, and restores geometrical shapes and mechanical properties of osteotomized bone. Seventeen female cynomolgus monkeys aged 18–19 years were allocated into three groups: control (CNT, n = 6), low-dose PTH (0.75 μg/kg; PTH-L, n = 6), and high-dose PTH (7.5 μg/kg; PTH-H, n = 5) groups. In all animals, twice a week subcutaneous injection was given for 3 weeks. Then fracture was produced surgically by transversely cutting the midshaft of the right femur and fixing with stainless plate. After fracture, intermittent PTH treatment was continued until sacrifice at 26 weeks after surgery. The femora were assessed by soft X-ray, three-point bending mechanical test, histomorphometry, and degree of mineralization in bone (DMB) measurement. Soft X-ray showed that complete bone union occurred in all groups, regardless of treatment. Ultimate stress and elastic modulus in fractured femur were significantly higher in PTH-H than in CNT. Total area and percent bone area of the femur were significantly lower in both PTH-L and PTH-H than in CNT. Callus porosity decreased dose-dependently following PTH treatment. Mean DMB of callus was significantly higher in PTH-H than in CNT or PTH-L. These results suggested that PTH decreased callus size and accelerated callus maturation in the fractured femora. PTH accelerates the natural fracture healing process by shrinking callus size and increasing degree of mineralization of the fracture callus, thereby restoring intrinsic material properties of osteotomized femur shaft in cynomolgus monkeys although there were no significant differences among the groups for structural parameters.

1α,25-Dihydroxy-2β(3-hydroxypropoxy)vitamin D 3 (ED-71) suppressed callus remodeling but did not interfere with fracture healing in rat femora

Introduction Because osteoporotic patients are prone to fractures, it must be considered whether or not patients undergoing drug therapies should discontinue treatment after sustaining a non-vertebral fracture. This study has tested the effect of novel active vitamin D 3 analog, 1α,25-dihydroxy-2β(3-hydroxypropoxy)vitamin D 3 (ED-71), on the fracture healing comparing with a powerful anti-resorptive agent, alendronate, using a rat femoral fracture model. Materials and methods Female SD rats ( n = 201) allocated into 6 groups were treated with MCT-vehicle and ED-71 at 0.025 and 0.05 μg/kg/day (EDL and EDH groups), and with saline-vehicle and alendronate at 5 and 10 μg/kg/day (ALL and ALH groups). After 4 weeks of pretreatment, osteotomy of the femur was performed. Treatment was continued until sacrifice at 6 and 16 weeks post-fracture. Fracture callus was evaluated by soft X-ray radiography, pQCT, biomechanical testing and histomorphometry. Results At 16 weeks post-fracture, new cortical shell appeared in 100% of Control (MCT and saline-vehicle), EDL and EHL, and in 67% and 56% of ALL and ALH, respectively. ED-71 treatment showed insignificantly large callus area only at 6 weeks, while alendronate treatment induced bigger callus at both 6 and 16 weeks post-fracture. The lamellar/callus area was decreased only at 6 weeks by ED-71 treatment, but both at 6 and 16 weeks by alendronate treatment. Osteoclast number in callus surface was decreased in both ED-71 and alendronate treatment groups at 6 weeks and in EDH, ALL and ALH at 16 weeks, indicating that ED-71 inhibits osteoclastic bone resorption, but its effect is less prominent than alendronate. Almost complete callus remodeling was observed in ED-71-treated groups at 16 weeks without any significant change in structural and material properties of fractured bone. Conclusions ED-71 suppression of callus remodeling by inhibiting osteoclastic bone resorption was mild and dose-dependent and did not interfere with natural fracture healing process at 16 weeks post-fracture.

Concepts of fracture union, delayed union, and nonunion.

Laboratory and clinical scientists and practicing clinicians need definitions of union, delayed union, and nonunion. Fracture union is a gradual process, so quantitative measures are the most meaningful. However, end point definitions also are useful, but they need empirical validation. The measure that has received the best validation in human fractures is bending stiffness. Quantitative radiologic assessment of healing is difficult because varying patterns of bone bridging can occur, including periosteal, endosteal, and intercortical patterns. Natural fracture healing was studied in 43 cases of isolated, closed, conservatively treated tibial shaft fracture with serial measurements of bending stiffness and standard radiographs. Three healing groups were defined on the basis of stiffness recovery patterns. Four cases showed delayed union, defined as failure to reach a stiffness of 7 N-m per degree by 20 weeks from fracture. The remaining cases had normal union, but at differing rates. Callus index was used as a measure of periosteal new bone formation. Stiffness measurements correlated more strongly than callus index with injury severity and functional outcome at 6 months. However, the callus index predicted behavior in those fractures that showed no tendency to heal at the 10-week stage. That is, absence of periosteal new bone in these cases presaged delayed union. These delayed union cases all eventually healed, still without producing periosteal callus, but other fractures in the series healed very rapidly, also without periosteal callus. The implication is that endosteal healing is capable of very rapid fracture bridging if conditions are right, but it also can occur late, after the periosteal healing response has ceased. These observations suggest a more rational approach to the definition of union, delayed union, and nonunion than that provided by the selection of arbitrary times. For conservatively treated fractures at least, delayed union can be defined as the cessation of the periosteal response before the fracture successfully has been bridged. Nonunion is the cessation of both the periosteal and endosteal healing responses without bridging.