Cortical Bone Healing Research Articles

CD11B + CD36 + cells are bone anabolic macrophages that limit age-associated bone loss.

Disruptions in the bone remodeling cycle that occur with increasing age lead to degeneration of the skeleton and increased risk of fragility fractures. Our understanding of how the bone remodeling process within cortical bone is controlled and altered with age in males and females is limited. Here, we generated bone marrow chimeric mice to understand the impacts of age and sex on the bone remodeling process. We demonstrate that transplantation of aged male or female bone marrow into young lethally irradiated male hosts unexpectedly enhances cortical bone mass without an impacting cancellous bone. Our single cell RNA-sequencing data show that mice reconstituted with aged bone marrow exhibited subsets of cells marked by CD11B/CD36 expression that demonstrate enhanced production of anabolic cytokines as compared to young counterparts, and that these myeloid subsets exist under conditions of normal physiology in aged mice. Importantly, CD11B + CD36 + cells do not differentiate into osteoclasts in vitro, and CD36 does not mark TRAP+ cells in vivo. Instead, CD36 + cells localize to resorption sites, including within cortical bone defects, suggesting their involvement in cortical bone remodeling and healing. CD11B + CD36 + cells also express elevated levels of bone anabolic WNT ligands, especially Wnt6. In functional assays, we demonstrate that soluble factors produced by CD11B + CD36 + cells enhance osteoblast progenitor commitment, mineralization, and activation of WNT signaling in vitro. Moreover, CD11B/CD36 exquisitely mark a subset of anabolic myeloid cells within human bone marrow. In conclusion, our studies identified a novel population of aged macrophages that limit cortical bone loss.

Delayed cortical bone healing due to impaired nuclear Nrf2 translocation in COPD mice

The effect of the pathogenesis of chronic obstructive pulmonary disease (COPD) on bone fracture healing is unknown. Oxidative stress has been implicated in the systemic complications of COPD, and decreased activity of Nrf2 signaling, a central component of the in vivo antioxidant mechanism, has been reported. We investigated the process of cortical bone repair in a mouse model of elastase-induced emphysema by creating a drill hole and focusing on Nrf2 and found that the amount of new bone in the drill hole was reduced and bone formation capacity was decreased in the model mice. Furthermore, nuclear Nrf2 expression in osteoblasts was reduced in model mice. Sulforaphane, an Nrf2 activator, improved delayed cortical bone healing in model mice. This study indicates that bone healing is delayed in COPD mice and that impaired nuclear translocation of Nrf2 is involved in delayed cortical bone healing, suggesting that Nrf2 may be a novel target for bone fracture treatment in COPD patients.

Osteogenic capacity of octacalcium phosphate involving macrophage polarization.

Previous research has found that octacalcium phosphate (OCP) increases macrophage accumulation and alters the initial inflammatory response. However, the role of the immune response induced by OCP in osteogenesis remains unknown. This study investigated the behavior of macrophages and bone regeneration capacity during the early inflammatory stage of OCP-mediated osteogenesis. To assess the change in macrophage polarization and osteogenic capacity, we used a standardized rat defect model filled with OCP or calcium-deficient hydroxyapatite (CDHA)-a material obtained through the hydrolysis of the original OCP. OCP or CDHA granules were incubated with RAW264 cells for 5 days to investigate the effect of physicochemical characteristics on macrophage cytokine/chemokine expression in vitro. Our in vivo results show that due to the OCP implantation, macrophages in the rat tibial defect area tend to polarize to the M2 phenotype (anti-inflammatory) and inhibit the formation of the M1 phenotype (pro-inflammatory). In comparison to CDHA, OCP exhibited superior bone regeneration potential due to its rapid promotion of cortical bone healing and stimulation of macrophage-related growth factors. Furthermore, our in vitro results have shown that OCP regulates the expression of macrophage chemokines over time. Compared to incubation with CDHA, incubation with OCP caused changes in the ionic microenvironment. These findings suggest that the OCP-mediated macrophage polarization and secretion profile not only regulate immune function but also positively affect osteogenesis.

Guided cortical and cancellous bone formation using a minimally invasive technique of BMSC- and BMP-2-laden visible light-cured carboxymethyl chitosan hydrogels

A cavity defect inside the bone is formed by deformed cancellous bone from the fixation of the cortical bone, and consequently, abnormal bone healing occurs. Therefore, repairing cancellous bone defects is a remarkable topic in orthopedic surgery. In this study, we prepared bone marrow-derived stem cell (BMSC)-laden and bone morphogenetic protein-2 (BMP-2)-laden visible light-cured carboxymethyl chitosan (CMCS) hydrogels for cortical and cancellous bone healing. Proton nuclear magnetic resonance (1H NMR) analysis confirmed the methacrylation of CMCS (CMCSMA), resulting in 55 % of substitution. The higher concentration of CMCSMA hydrogel resulted in the lower swelling ratio, the larger viscosity, the slower degradation behavior, and the stronger compressive strength. The 5 w/v% hydrogel exhibited a controlled BMP-2 release for 14 days, while the 7 and 10 w/v% hydrogels displayed a controlled BMP-2 release for 28 days. Results of in vitro cytotoxicity and cell proliferation assays revealed the biocompatibility of the samples. In vivo animal tests demonstrated that BMSC- and BMP-2-laden 7 w/v% CMCSMA (CMCSMA+Cell+BMP-2) improved bone formation in the defected cortical and cancellous bones of the femur, as analyzed by micro-computed tomography (micro-CT) and histological evaluations. Consequently, we suggested that CMCSMA+Cell+BMP-2 can be a valuable scaffold for restoring cortical and cancellous bone defects.

Recycled windshield glass as new material for producing ultrasonic phantoms of cortical bone-healing stages

The quantitative ultrasound technique was used to evaluate bone-mimicking phantoms; however, these phantoms do not mimic the intermediate stages of cortical bone healing. We propose using windshield glass as an original material to produce phantoms that mimic the characteristics of three different stages of cortical bone healing. This material was processed via a route that included breaking, grinding, compacting, drying, and sintering in four temperature groups: 625 °C, 645 °C, 657 °C, and 663 °C. The parameters evaluated were the ultrasonic longitudinal phase velocity (), corrected () ultrasonic attenuation coefficient, and bulk density (). The results showed that the mean values of and varied from 2, 398 to 4, 406 m·s−1, 3 to 10 dB·cm−1, and 1, 563 to 2, 089 kg·m−3, respectively. The phantoms exhibited properties comparable with the three stages of cortical bone healing and can be employed in diagnostic and therapeutic studies using ultrasound.

The Evaluation of Different Osteotomy Drilling Speed Protocols on Cortical Bone Temperature, Implant Stability and Bone Healing: An Experimental Study in an Animal Model.

The aim of this study was to measure the effect of drilling speed on heat generation in the cortical bone, on primary and secondary implant stability of implants and on early and late bone healing with micro-computerized tomography (micro-CT). Sixty implants were placed in the iliac crest of 6 sheep in order to form 5 different drilling protocols: 50 rpm without saline cooling, and 400, 800, 1200, and 2000 rpm with saline cooling. Simultaneous cortical bone temperature and primary stability at the time of placement; secondary stability and the ratio between relative bone and tissue volume (BV/TV) in 2D and 3D in micro-CT analysis were evaluated after 4 and 8 weeks. The 50-rpm group had the highest cortical bone temperature and the longest operation duration with the highest primary stability. Slightly higher values of secondary stability (T2) and subsequent 2D and 3D BV/TV values were found in 1200 rpm with irrigation at 8 weeks. All groups had sufficient ISQ values at 8 weeks for loading although the micro-CT analysis showed varying percentages of bone tissue around implants. The influence of drill speed for implant osteotomy and its irrigation is minimal when it comes to changes in temperature of the cortical bone, primary and secondary implant stability, and BV/TV.

Collagen Membrane for Guided Bone Regeneration in Dental and Orthopedic Applications.

Treatment of cortical bone defects is a clinical challenge. Guided bone regeneration (GBR), commonly used in oral and maxillofacial dental surgery, may show promise for orthopedic applications in repair of cortical bone defects. However, a limitation in the use of GBR for cortical bone defects is the lack of an ideal scaffold that provides sufficient mechanical support to bridge the cortical bone with minimal interference in the repair process. We have developed a new collagen membrane, CelGro™, for use in GBR. We report the material characterization of CelGro and evaluate the performance of CelGro in translational preclinical and clinical studies. The results show CelGro has a bilayer structure of different fiber alignment and is composed almost exclusively of type I collagen. CelGro was found to be completely acellular and free from xenoantigen, α-gal (galactose-alpha-1,3-galactose). In the preclinical study of a rabbit cortical bone defect model, CelGro demonstrated enhanced bone-remodeling activity and cortical bone healing. Microcomputed tomography evaluation showed early bony bridging over the defect area 30 days postoperatively, and nearly complete restoration of mature cortical bone at the bone defect site 60 days postoperatively. Histological analysis 60 days after surgery further confirmed that CelGro enables bridging of the cortical bone defect by induction of newly formed cortical bone. Compared to a commercially available collagen membrane, Bio-Gide®, CelGro showed much better cortical alignment and reduced porosity at the defect interface. As selection of orthopedic patients with cortical bone defects is complex, we conducted a clinical study evaluating the performance of CelGro in guided bone regeneration around dental implants. CelGro was used in GBR procedures in a total of 16 implants placed in 10 participants. Cone-beam computed tomography images show significantly increased bone formation both horizontally and vertically, which provides sufficient support to stabilize implants within 4 months. Together, the findings of our study demonstrate that CelGro is an ideal membrane for GBR not only in oral and maxillofacial reconstructive surgery but also in orthopedic applications (Clinical Trial ID ACTRN12615000027516).

The effects of repeated usage of implant drills on cortical bone temperature, primary/secondary stability and bone healing: A preclinical in vivo micro-CT study.

The aims of the study were to evaluate the effects of new vs. used drills on cortical bone temperature, operation time, and primary/secondary stability and to assess bone healing by micro-CT. New, 50×-used and 100×-used drills at 800rpm were used to place implants in the iliac crests of three male sheep in this experimental in vivo animal study. A total of 36 implants (12 in each group) were placed. The temperature of the cortical bone was recorded with an infrared thermometer, and the ISQ values at implant placement (primary stability) and at 4weeks (secondary stability) were evaluated. The bone volume assessed by micro-CT was used to evaluate bone tissue healing. The results were statistically compared. The results have shown that the operation time and the cortical bone temperature increased by the usage of the drills (p<.05) and the primary/secondary stability (ISQ values) decreased with the number of drill usage (p<.05) The highest 2- and 3-dimensional bone volume (assessed by micro-CT) were noted for new drills group followed by 50×-used drills and 100×-used drills (p<.05 at 4weeks). The number of drill usage seems to be directly affecting the osseointegration process in implant surgery. Therefore, replacing the drills at certain time intervals may increase the success of implant treatment.

The combined effects of teriparatide and anti-RANKL monoclonal antibody on bone defect regeneration in ovariectomized mice.

The purpose of this study was to investigate the combined effects of teriparatide (TPTD) and anti-murine receptor activator of nuclear factor-κB ligand monoclonal antibody (anti-RANKL Ab) on both cancellous and cortical bone healing in ovariectomized mice. Thirteen-week-old mice were divided into the sham-operated group (n=11) or the ovariectomized group (n=44). At 1 month post-operation, all mice underwent bone defect surgery on the left femoral metaphysis (cancellous bone healing model) and right femoral mid-diaphysis (cortical bone healing model). After creating the bone defects, all ovariectomized mice were assigned to one of four groups to receive 1) saline (5 times a week; CNT group), 2) TPTD (40μg/kg 5 times a week; TPTD group), 3) anti-RANKL Ab (5mg/kg once; Ab group), or 4) a combination of TPTD and anti-RANKL Ab (COMB group). The following analyses were performed: Time-course microstructural analysis of healing in both cancellous and cortical bone in the bone defect, measuring the volumetric bone mineral density and the cortical bone thickness of the tibia as a representative of whole body bone with the use of micro-computed tomography, and histological analysis. Regeneration of cancellous bone volume in the COMB group was the highest among the four groups, and combined treatment accelerated the formation of medullary callus during the early phase of bone regeneration. On the other hand, there were no significant differences in the regeneration of cortical bone volume during the early phase of bone regeneration among the four groups. Furthermore, lamellar bone was not well identified in the all four groups. Volumetric bone mineral density in the tibia in the COMB group was significantly higher compared with that in the CNT and TPTD groups and tended to be higher compared with that in the Ab group. The mean values of cortical bone thickness in the TPTD and COMB groups were significantly higher than that in the CNT group. In a mouse model of postmenopausal osteoporosis, combination therapy of TPTD and anti-RANKL Ab accelerates regeneration of cancellous bone more effectively than either agent alone during the early phase of bone regeneration.

Different effects of Wnt/β-catenin activation and parathyroid hormone on diaphyseal and metaphyseal in the early phase of femur bone healing of mice.

SummaryParathyroid hormone (PTH) and agents related to the manipulation of Wnt/β‐catenin signalling are two promising anabolic anti‐osteoporotic therapies that have been shown to promote the healing of bone fractures. Now, it is widely accepted that cortical bone and trabecular bone are two different compartments, and should be treated as separate compartments in pathological processes, such as fracture healing. It is currently unknown whether PTH and the activation of β‐catenin signalling would demonstrate different effects on cortical bone and trabecular bone healing. In the current study, single 0.6‐mm cortex holes were made in the femur metaphysis and diaphysis of mice, and then, PTH application and β‐catenin activation were used to observe the promoting effect on bone healing. The effects of β‐catenin and PTH signalling on fracture healing were observed by X‐ray and CT at 3, 6, and 14 days after fracture, and the levels of β‐catenin were detected by RT‐PCR assay, and the number of specific antigen‐positive cells of BRDU, OCN, RUNX2 was counted by immunohistochemical staining. While β‐catenin activation and PTH were found to demonstrate similar effects on accelerating metaphyseal bone healing, activation of β‐catenin showed a more striking effect than PTH on promoting diaphyseal bone healing. These findings might be helpful for selecting proper medication to accelerate fracture healing of different bone compartments.

Different composition of leucocytes in cortical and cancellous bone healing in a mouse model.

ObjectivesCortical and cancellous bone healing processes appear to be histologically different. They also respond differently to anti-inflammatory agents. We investigated whether the leucocyte composition on days 3 and 5 after cortical and cancellous injuries to bone was different, and compared changes over time using day 3 as the baseline.MethodsTen-week-old male C56/Bl6J mice were randomized to either cancellous injury in the proximal tibia or cortical injury in the femoral diaphysis. Regenerating tissues were analyzed with flow cytometry at days 3 and 5, using panels with 15 antibodies for common macrophage and lymphocyte markers. The cellular response from day 3 to 5 was compared in order to identify differences in how cancellous and cortical bone healing develop.ResultsBetween day 3 and 5, the granulocytes increased in the cancellous model, whereas the lymphocytes (T cells, B cells, NK cells) and monocytes (CD11b+, F4/80+, CD206+, CD14+) increased in the cortical model.ConclusionThese results suggest an acute type of inflammation in cancellous bone healing, and a more chronic inflammation in cortical healing. This might explain, in part, why cancellous healing is faster and more resistant to anti-inflammatory drugs than are diaphyseal fractures.Cite this article: L. Tätting, O. Sandberg, M. Bernhardsson, J. Ernerudh, P. Aspenberg. Different composition of leucocytes in cortical and cancellous bone healing in a mouse model. Bone Joint Res 2018;7:620–628. DOI: 10.1302/2046-3758.712.BJR-2017-0366.R2.

Guided tissue engineering for healing of cancellous and cortical bone using a combination of biomaterial based scaffolding and local bone active molecule delivery

A metaphyseal bone defect due to infection, tumor or fracture leads to loss of cancellous and cortical bone. An animal model separating the cancellous and cortical healing was used with a combination of a macroporous gelatin-calcium sulphate-hydroxyapatite (Gel-CaS-HA) biomaterial as a cancellous defect filler, and a thin collagen membrane (CM) guiding cortical bone regeneration. The membrane was immobilized with bone morphogenic protein-2 (rhBMP-2) to enhance the osteoinductive properties. The Gel-CaS-HA cancellous defect filler contained both rhBMP-2 and a bisphosphonate, (zoledronate = ZA) to prevent premature callus resorption induced by the pro-osteoclast effect of rhBMP-2 alone. In the first part of the study, the CM delivering both rhBMP-2 and ZA was tested in a muscle pouch model in rats and the co-delivery of rhBMP-2 and ZA via the CM resulted in higher amounts of bone compared to rhBMP-2 alone. Secondly, an established tibia defect model in rats was used to study cortical and cancellous bone regeneration. The defect was left empty, filled with Gel-CaS-HA alone, Gel-CaS-HA immobilized with ZA or Gel-CaS-HA immobilized with rhBMP-2+ZA. Functionalization of the Gel-CaS-HA scaffold with bioactive molecules produced significantly more bone in the cancellous defect and its surroundings but cortical defect healing was delayed likely due to the protrusion of the Gel-CaS-HA into the cortical bone. To guide cortical regeneration, the cortical defect was sealed endosteally by a CM with or without rhBMP-2. Subsequently, the cancellous defect was filled with Gel-CaS-HA containing ZA and rhBMP-2+ZA. In the groups where the CM was doped with rhBMP-2, significantly higher number of cortices bridged. The approach to guide cancellous as well as cortical bone regeneration separately in a metaphyseal defect using two bioactive molecule immobilized biomaterials is promising and could improve the clinical care of patients with metaphyseal defects.

Oncologic doses of zoledronic acid induce site specific suppression of bone modelling in rice rats.

To examine the effect of zoledronic acid (ZOL) on cortical bone modelling and healing of extraction sockets in the jaw bones of a rodent model. We hypothesized ZOL suppresses both the bone formation in the modelling mode in the jaw bones and alters the extraction site healing. Rice rats were administered saline solution and two dose regimens of ZOL: 0.1mg/kg, twice a week, for 4weeks (n=17, saline=8 & ZOL=9) and a higher dose of 0.4mg/kg, weekly, for 9weeks (n=30, saline=15 & ZOL=15). Two pairs of fluorochrome bone labels were administered. Extraction of maxillary teeth was performed in maxilla. Mineral apposition rate, mineralizing surface and bone formation rate (BFR) were quantified on periodontal (PDL), alveolar and basal bone surfaces, and in the trabecular bone of proximal tibia. Bone volume (BV) was evaluated at extraction sockets. Multivariate Gaussian models were used to account for repeated measurements, and analyzes were conducted in SAS V9.3. ZOL suppressed bone modelling (BFR/BS) at the PDL surfaces in the mandible (P<.05), but its effect was not significant at the periosteal surfaces of both jaws. BV for the healing sockets of ZOL treated animals was not significantly different (P=.07) compared to the saline group. ZOL suppressive effect was higher in the tibia compared to the jaws. ZOL severely suppresses coupled remodelling in the tibia, and the suppression of bone formation in the modelling mode in the jaws demonstrates the site specific effects of ZOL in rice rats.

Cortical and Trabecular Bone Healing Patterns and Quantification for Three Different Dental Implant Systems.

The present study hypothesized that different bone healing patterns through initial stages of osseointegration would be observed when three distinct commercially available implant systems (Nobel Groovy, Implacil, and Zimmer TSV) were used, leading to significant variations in histometric levels of total bone and new bone formation during the osseointegration process. A total of 48 implants were placed bilaterally on the tibias of eight beagle dogs and allowed to heal for 2 and 6 weeks. Following euthanasia, nondecalcified specimens were processed for morphologic and histometric evaluation. Bone-to-implant contact (BIC) and new bone area fraction occupancy (BAFO) analyses for native and new bone were performed along the whole perimeter of each implant and separately for the cortical and trabecular bone regions. Morphologic evaluation of cortical bone presented different healing patterns and osseointegration levels for different implant systems as time elapsed in vivo. Interfacial remodeling was the chief healing pattern in Zimmer implants, while a combination of interfacial remodeling and healing chambers was observed in Nobel and Implacil implants. When trabecular bone was evaluated, similar bone healing patterns were observed between systems despite different levels of osseointegration observed as a function of implantation time, implant system, and native and/or new bone BIC and BAFO. Different implant systems led to different healing patterns during early stages of osseointegration. Such variation in pattern was more noticeable in the cortical regions compared to the trabecular regions. The variation in bone healing pattern did significantly influence overall indicators of native and new BIC and BAFO during the osseointegration process. The postulated hypothesis was accepted.

Compressive mechanical properties of the radial bone defects treated with nano-hydroxyapatite/yttria-stabilized zirconia in rabbits

The treatment of massive bone defects is one of the most challenging problems, and many researchers are trying to find materials or drugs that can improve bone healing. The aim of this study was to evaluate the biomechanical effect of nano-hydroxyapatite/yttria-stabilized zirconia on the healing of a radial cortical bone defect in a rabbit model. Thirty six adult male New Zealand rabbits were randomly allocated into two equal groups and three subgroups (1-, 2-, and 3-month duration) with six rabbits in each subgroup. Under general anesthesia, a segmental bone defect, 10 mm in length, was created in the middle of the right radial shaft in all rabbits. This was then stabilized using mini-plate with four screws. The defects in group I were left empty, whereas in group II, the bone defects were filled with nano-hydroxyapatite/yttria-stabilized zirconia. The mean load for fracturing radial bone in group II at 1, 2, and 3 months after surgery was significantly higher than group I (P < 0.05). Biomechanical analysis of the radial bone healing was shown to be significantly improved by the nano-hydroxyapatite/yttria-stabilized zirconia compared with the control group, suggesting that this material promotes the healing of cortical bone, presumably by acting as an osteoconductive agent.

Study of nano-hydroxyapatite/zirconia stabilized with yttria in bone healing: histopathological study in rabbit model.

Acceleration of bone healing has always been a major challenge in orthopedic surgery, the aim of this study was an evaluation of the biological effects of zirconia-stabilized yttria on bone healing, using an in vivo model. Nano-hydroxyapatite powder with zirconia-stabilized yttria were inserted in rabbit tibia and then histologically analyzed and compared with non-treated controls so thirty six. New Zealand white male rabbits randomly divided into two groups of 18 rabbits each. A cortical hole of 4 mm diameter and 8 mm depth in each tibia was drilled. In group I, the defect was left empty, whereas in group II, the bone defect was packed with nano-hydroxyapatite/5% zirconia stabilized with yttria. Histological evaluations were performed at two, four and six weeks after the implantation. Microscopic changes on two groups along with the time course were scored and statistical analysis showed that the average scores in group II were significantly higher than the other groups (p < 0.05). Histological analysis was shown to be significantly improved by the nano-hydroxyapatite/5% zirconia stabilized with yttria compared with the control group, suggesting that this biomaterial promote the healing of cortical bone, presumably by acting as an osteoconductive.

Vertical bone augmentation procedures: Basics and techniques in dental implantology

An appropriate bony situation is essential for dental implant placement and bony support of soft tissues (pink esthetic). Loss of teeth often results in complex horizontal and vertical alveolar ridge defects. They demand advanced bone augmentation techniques for reconstruction. We present the different techniques and materials used in complex bone augmentation. Clinical cases show the application of the methods in the clinical setting. We present current techniques and materials used in complex bone augmentations. Clinical cases show the application of the methods in the clinical setting. Applied techniques include stabilized-guided bone regeneration (GBR), autologous local block augmentation, modified techniques such as Gellrich shell technique including piezosurgery, pelvic bone blocks, complex materials such as graft-derived bone blocks and their unique handling problems. Successful basic principles are reduction of cortical bone healing due to long remodeling time and possible late loss; extended application of materials with interconnecting porous system and particulate material resulting in fast healing analogous to cancellous bone; mechanical stabilization of the augmentation to allow bony healing in vertical defect situations. GBR and autologous bone blocks with minimal cortical thickness and a high volume of particulated material are most favorable techniques.

Combination of poly L-lactic acid nanofiber scaffold with omentum graft for bone healing in experimental defect in tibia of rabbits

To investigate the osteoconductive properties and biological performance of Poly L-lactic acid (PLLA) with omentum in bone defects. PLLA nanofiber scaffolds were prepared via electrospinning technique. Forty four New Zealand white female rabbits randomly divided into three groups of 18 rabbits each. Created defects in right tibias were filled in group I with omentum, in group II with PLLA nanofiber scaffold and in group III with combination of the omentum and PLLA. The same defects were created in left tibia of all groups but did not receive any treatment (control group). Histological and histomorphometric evaluations were performed at two, four and six weeks after the implantation. Histological changes on all groups along with the time course were scored and statistical analysis showed that the average scores in group III were significantly higher than the other groups. Histomorphometric analysis of bone healing was shown to be significantly improved by the combined PLLA with omentum compared with the other groups, suggesting this biomaterial promote the healing of cortical bone, presumably by acting as an osteoconductive scaffold.

Impaired bone healing pattern in mice with ovariectomy-induced osteoporosis: A drill-hole defect model

To establish a drill-hole defect model in osteoporotic mouse femur by comparing temporal cortical bone healing pattern between OVX-induced osteoporotic bone and sham-operated bone. 3-month-old female C57BL/6 mice were randomly divided into an ovariectomy group (OVX) and a sham-operated group (Sham). At 6 weeks post-surgery, 7 mice from each group were sacrificed to examine the distal femur and femoral shaft by both micro-CT and mechanical testing for confirming established osteoporosis induced by OVX. In the remaining mice, a cortical bone defect 0.8mm in diameter was created on the mid-diaphysis of the right femur. The local repair process at days 0, 3, 7, 10, 14 and 21 after creation of the drill-hole was in vivo monitored by high-resolution micro-CT scanning. At each time point, each animal was scanned four times and was removed from the scanner between scans to determine reproducibility. Mice were sacrificed at each time point (n=12 at days 0, 3, 7, 10 and 14; n=20 at day 21). Before sacrifice, sera were collected to examine expression of bone formation marker P1NP (procollagen type I N-terminal propeptide) and bone resorption marker CTX (C-terminal telopeptide of type I collagen). After sacrifice, callus samples were collected and subjected to the following analyses: micro-CT-based angiography; histological examination; immunohistochemical staining to determine estrogen receptor expression; quantitative real-time PCR analysis of collagen type I, collagen type II, collagen type X, osteocalcin, tartrate-resistant acid phosphatase, estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta) gene expression; and three-point mechanical testing. At 6 weeks post-surgery, OVX mice had significantly lower bone mass, impaired bone micro architecture and compromised mechanical properties compared to the Sham mice. In vivo micro-CT analysis revealed that the bone volume fraction in the defect region was significantly lower in the OVX group from day 10 to day 21 post-injury as compared to the Sham group, and was significantly lower in the intra-medulla region in the OVX group from day 7 to day 14 as compared to the Sham group, consistent with the histological data. Analysis of bone biochemical markers indicated that circulating P1NP levels normalized by baseline in the OVX mice were significantly lower than in the Sham mice from day 7 to day 10, and that temporal expression of circulating CTX levels normalized by baseline was also lower in the OVX mice as compared to the Sham mice. These results were consistent with quantitative real-time PCR analysis. ER alpha mRNA expression was significantly lower in the OVX mice, whereas ER beta mRNA expression was significantly higher in the OVX mice as compared to the Sham mice at all time points examined, consistent with immunohistochemical staining. The restoration of femoral mechanical property, determined based on ultimate load and energy-to-failure, was significantly lower in the OVX mice than in the Sham mice. In addition, in vivo micro-CT scanning for quantifying new bone formation in the defect site was highly reproducible in this model. The bone healing of the drill-hole defect was impaired in mice with OVX-induced osteoporosis. The present study provides a model to investigate the functional role of specific gene in osteoporotic bone healing and may facilitate development of novel therapeutic strategies for promoting osteoporotic bone healing.

Characterizing gait induced normal strains in a murine tibia cortical bone defect model

The critical role that mechanical stimuli serve in mediating bone repair is recognized but incompletely understood. Further, previous attempts to understand this role have utilized application of externally applied mechanical loads to study the tissue’s response. In this project, we have therefore endeavored to capitalize on bone’s own consistently diverse loading environment to develop a novel model that would enable assessment of the influence of physiologically engendered mechanical stimuli on cortical defect repair. We used an inverse dynamics approach with finite element analysis (FEA) to first quantify normal strain distributions generated in mouse tibia during locomotion. The strain environment of the tibia, as previously reported for other long bones, was found to arise primarily due to bending and was consistent in orientation through the stance phase of gait. Based on these data, we identified three regions within a transverse cross-section of the mid-diaphysis as uniform locations of either peak tension, peak compression, or the neutral axis of bending (i.e. minimal strain magnitude). We then used FEA to quantify the altered strain environment that would be produced by a 0.6mm diameter cylindrical cortical bone defect at each diaphyseal site and, in an in situ study confirmed our ability to accurately place defects at the desired diaphyseal locations. The resulting model will enable the exploration of cortical bone healing within the context of physiologically engendered mechanical strain.