Dick Ho Kiu Chow

Implant-Derived Magnesium Induces Local Neuronal Production of CGRP to Improve Bone-Fracture Healing in Rats

Orthopedic implants containing biodegradable magnesium have been used for fracture repair with considerable efficacy; however, the underlying mechanisms by which these implants improve fracture healing remain elusive. Here we show the formation of abundant new bone at peripheral cortical sites after intramedullary implantation of a pin containing ultrapure magnesium into the intact distal femur in rats. This response was accompanied by substantial increases of neuronal calcitonin gene-related polypeptide-α (CGRP) in both the peripheral cortex of the femur and the ipsilateral dorsal root ganglia (DRG). Surgical removal of the periosteum, capsaicin denervation of sensory nerves or knockdown in vivo of the CGRP-receptor-encoding genes Calcrl or Ramp1 substantially reversed the magnesium-induced osteogenesis that we observed in this model. Overexpression of these genes, however, enhanced magnesium-induced osteogenesis. We further found that an elevation of extracellular magnesium induces magnesium transporter 1 (MAGT1)-dependent and transient receptor potential cation channel, subfamily M, member 7 (TRPM7)-dependent magnesium entry, as well as an increase in intracellular adenosine triphosphate (ATP) and the accumulation of terminal synaptic vesicles in isolated rat DRG neurons. In isolated rat periosteum-derived stem cells, CGRP induces CALCRL- and RAMP1-dependent activation of cAMP-responsive element binding protein 1 (CREB1) and SP7 (also known as osterix), and thus enhances osteogenic differentiation of these stem cells. Furthermore, we have developed an innovative, magnesium-containing intramedullary nail that facilitates femur fracture repair in rats with ovariectomy-induced osteoporosis. Taken together, these findings reveal a previously undefined role of magnesium in promoting CGRP-mediated osteogenic differentiation, which suggests the therapeutic potential of this ion in orthopedics.

Low-magnitude high-frequency vibration (LMHFV) enhances bone remodeling in osteoporotic rat femoral fracture healing.

Low‐magnitude high‐frequency vibration (LMHFV) (35 Hz, 0.3 g) accelerates fracture healing by enhancing callus formation and mineralization for both normal and osteoporotic rats in our previous studies. 1,2 We hypothesized that LMHFV enhances fracture healing through bone remodeling. Ibandronate was used to suppress LMHFV‐stimulated bone remodeling and changes in remodeling were investigated to verify our hypothesis. Closed femoral fractures were created in 80 osteoporotic female Sprague–Dawley rats. The rats were randomly assigned into control (CG), LMHFV (VG) (20 min/day, 5 days/week), ibandronate (BG) (7 µg/kg/week), or LMHFV + ibandronate (VBG) for a treatment duration of 2, 4, 6, or 8 weeks. Blood was taken and the femora were harvested for histological and radiological analyses. VG had the fastest drop in callus area (CA) and width (CW), and bone volume to tissue volume ratio (BV/TV); whereas, a plateaued trend in BG and VBG was observed. The fastest callus reduction, highest mineral apposition rate at week 6, and increased serum concentration of osteocalcin and TRAP5b in VG suggested enhanced remodeling. LMHFV partially reversed the inhibition of bone remodeling by ibandronate suggested LMHFV had an opposite effect on bone remodeling to ibandronate. In conclusion, LMHFV accelerated fracture healing by enhancing bone remodeling and the administration of ibandronate can impair this enhancement. LMHFV has great potential in improving fracture outcome clinically.

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.

Sclerostin Antibody Treatment Increases Bone Formation, Bone Mass, and Bone Strength of Intact Bones in Adult Male Rats.

We investigated the systemic effect of sclerostin monoclonal antibody (Scl-Ab) treatment on intact non-operated bones in an open osteotomy male Sprague Dawley (SD) rat model. Six-month-old male SD rats were subjected to transverse osteotomy at the right femur mid-shaft. Rats were injected subcutaneously with vehicle or Scl-Ab (25 mg/kg, 2 times per week) treatment for 9 weeks. Compared with vehicle control, Scl-Ab treatment significantly improved trabecular and cortical bone mass and microarchitecture at L5 vertebrae and left femora by micro-CT at week 6 and 9. Mechanical testing showed that Scl-Ab treatment resulted in significantly higher stiffness, energy to failure and ultimate load at the femora at week 9. Mineral apposition rate, mineralizing surface and bone formation rate on the trabecular bone in the distal femora was significantly increased in Scl-Ab group at week 6 and 9. The administered Scl-Ab was localized in the osteocytes and beta-catenin was strongly expressed in osteoblasts. Scl-Ab treatment significantly increased serum P1NP level and there was no between-group difference in serum level of CTX-1. In conclusion, Scl-Ab treatment could induce rapid and sustained increase in bone formation, bone mass and bone strength in non-operated bones. Sclerostin inhibition might be advantageous to prevent secondary fracture(s).

Extracorporeal Shockwave Therapy for Treatment of Delayed Tendon-Bone Insertion Healing in a Rabbit Model A Dose-Response Study

Background: Tendon–bone insertion (TBI) consists of both hard and soft tissues. TBI injury with delayed repair is not uncommon. High-dose extracorporeal shockwave (ESW) is effective for treating nonunion fracture, whereas low-dose ESW is used for tendinopathy therapy. The dosing effect of ESW on delayed TBI healing is lacking. Hypothesis: Low-dose ESW might have a healing enhancement effect comparable to that of high-dose ESW in treating delayed TBI healing. Study Design: Controlled laboratory study. Methods: Partial patellectomy was adopted to create a delayed TBI healing model by shielding the healing interface between tendon and bone. Ninety-six female New Zealand White rabbits with unilateral delayed TBI healing at the knee joint were divided into 3 groups: controls, low-dose ESW (LD-ESW; 0.06 mJ/mm2, 4 Hz, 1500 impulses), and high-dose ESW (HD-ESW; 0.43 mJ/mm2, 4 Hz, 1500 impulses). The TBI shielding was removed at week 4 after partial patellectomy, followed by treatment with control or ESW at week 6. The rabbits were euthanized at week 8 and week 12 for radiological, microarchitectural, histological, and mechanical assessments of healing tissues. Results: Radiologically, both the LD-ESW group and the HD-ESW group showed larger new bone area than the controls at week 8 and week 12. Microarchitectural measurements showed that the LD-ESW and HD-ESW groups had larger new bone volume than the controls at week 12. Histological assessments confirmed osteogenesis enhancement. Both the LD-ESW and HD-ESW groups showed significantly higher failure load at the TBI healing complex than the control group at week 12. No significant difference was detected between the 2 ESW treatment groups at week 8 or week 12. Conclusion: Extracorporeal shockwave, a unique noninvasive physical modality, had similar effects between the low and high dose for treating delayed TBI healing. Clinical Relevance: Low-dose ESW for TBI delayed healing might be more desirable and have better compliance in clinical applications.

Blockage of Src by Specific siRNA as a Novel Therapeutic Strategy to Prevent Destructive Repair in Steroid-Associated Osteonecrosis in Rabbits

Vascular hyperpermeability and highly upregulated bone resorption in the destructive repair progress of steroid‐associated osteonecrosis (SAON) are associated with a high expression of VEGF and high Src activity (Src is encoded by the cellular sarcoma [c‐src] gene). This study was designed to prove our hypothesis that blocking the VEGF‐Src signaling pathway by specific Src siRNA is able to prevent destructive repair in a SAON rabbit model. Destructive repair in SAON was induced in rabbits. At 2, 4, and 6 weeks after SAON induction, VEGF, anti‐VEGF, Src siRNA, Src siRNA+VEGF, control siRNA, and saline were introduced via intramedullary injection into proximal femora for each group, respectively. Vascularization and permeability were quantified by dynamic contrast‐enhanced (DCE) MRI. At week 6 after SAON induction, proximal femurs were dissected for micro–computed tomography (μCT)‐based trabecular architecture with finite element analysis (FEA), μCT‐based angiography, and histological analysis. Histological evaluation revealed that VEGF enhanced destructive repair, whereas anti‐VEGF prevented destructive repair and Src siRNA and Src siRNA+VEGF prevented destructive repair and enhanced reparative osteogenesis. Findings of angiography and histomorphometry were consistent with those determined by DCE MRI. Src siRNA inhibited VEGF‐mediated vascular hyperpermeability but preserved VEGF‐induced neovascularization. Bone resorption was enhanced in the VEGF group and inhibited in the anti‐VEGF, Src siRNA, Src siRNA+VEGF groups as determined by both 3D μCT and 2D histomorphometry. FEA showed higher estimated failure load in the Src siRNA and Src siRNA+VEGF groups when compared to the vehicle control group. Blockage of VEGF‐Src signaling pathway by specific Src siRNA was able to prevent steroid‐associated destructive repair while improving reconstructive repair in SAON, which might become a novel therapeutic strategy.

A Comparative Study on the Biomechanical and Histological Properties of Bone-to-Bone, Bone-to-Tendon, and Tendon-to-Tendon Healing: An Achilles Tendon–Calcaneus Model in Goats

Background: Surgical repair around the bone-tendon insertion (BTI) may involve bone-to-bone (BB), bone-to-tendon (BT), or tendon-to-tendon (TT) reattachment with varying healing outcome. Hypothesis: The repair of Achilles tendon–calcaneus (ATC) by reattachment of homogeneous tissue (BB or TT) would heal faster, with respect to tensile properties at the healing complex, than those of reattachment of heterogeneous tissues (BT) over time. Study Design: Controlled laboratory study. Methods: Forty-seven adolescent male Chinese goats were divided into BB, BT, and TT groups. Osteotomy of the calcaneus, reattachment of Achilles tendon to the calcaneus after removal of the insertion, and tenotomy of the Achilles tendon were performed to simulate BB, BT, and TT repair, respectively. The ATC healing complexes were harvested at 6, 12, or 24 weeks postoperatively. Mechanical and morphological properties of the healing ATC complexes were assessed by tensile testing and qualitative histology, respectively. The contralateral intact ATC complex was used as the control. Results: Failure load of BT was 33.4% lower than that of TT (P = .0243) at week 12. Ultimate strength of BT was 50.2% and 45.3% lower than that of TT at weeks 12 (P = .0002) and 24 (P = .0001), respectively. Tissue morphological characteristics of the BB and TT groups showed faster remodeling. The BT group showed limited regeneration of fibrocartilage zone and excessive formation of fibrous tissue at the healing interface. Conclusion: BTI repair between homogeneous tissues (BB and TT healing) showed better healing quality with respect to mechanical and histological assessments than did healing between heterogeneous tissues (BT healing). Clinical Relevance: Anatomic reconstruction of ATC complex injury may be a primary concern when selecting the proper surgical approach. However, it is recommended to select fracture fixation (BB) or tendon repair (TT) instead of bone-tendon reattachment (BT) if possible to ensure better outcome at the healing interface.

Extracorporeal shockwave enhanced regeneration of fibrocartilage in a delayed tendon-bone insertion repair model

Fibrous tissue is often formed in delayed healing of tendon bone insertion (TBI) instead of fibrocartilage. Extracorporeal shockwave (ESW) provides mechanical cues and upregulates expression of fibrocartilage‐related makers and cytokines. We hypothesized that ESW would accelerate fibrocartilage regeneration at the healing interface in a delayed TBI healing model. Partial patellectomy with shielding at the TBI interface was performed on 32 female New Zealand White Rabbits for establishing this delayed TBI healing model. The rabbits were separated into the control and ESW group for evaluations at postoperative week 8 and 12. Shielding was removed at week 4 and a single ESW treatment was applied at week 6. Fibrocartilage regeneration was evaluated histomorphologically and immunohistochemically. Vickers hardness of the TBI matrix was measured by micro‐indentation. ESW group showed higher fibrocartilage area, thickness, and proteoglycan deposition than the control in week 8 and 12. ESW increased expression of SOX9 and collagen II significantly in week 8 and 12, respectively. ESW group showed a gradual transition of hardness from bone to fibrocartilage to tendon, and had a higher Vickers hardness than the control group at week 12. In conclusion, ESW enhanced fibrocartilage regeneration at the healing interface in a delayed TBI healing model.

Magnesium (Mg) based interference screws developed for promoting tendon graft incorporation in bone tunnel in rabbits

How to enhance tendon graft incorporation into bone tunnels for achieving satisfactory healing outcomes in patients with anterior cruciate ligament reconstruction (ACLR) is one of the most challenging clinical problems in orthopaedic sports medicine. Several studies have recently reported the beneficial effects of Mg implants in bone fracture healing, indicating the use potential of Mg devices in promoting the tendon graft osteointegration. Here, we developed an innovative Mg-based interference screws for fixation of the tendon graft in rabbits underwent ACLR and investigated the biological role of Mg-based implants in the graft healing. The titanium (Ti) interference screw was used as the control. We demonstrated that Mg interference screw significantly accelerated the incorporation of the tendon graft into bone tunnels via multiscale analytical methods including scanning electronic microscopy/energy dispersive spectrometer (SEM/EDS), micro-hardness, micro-Fourier transform infrared spectroscopy (μFTIR), and histology. Our in vivo study showed that Mg implants enhanced the recruitment of bone marrow stromal stem cells (BMSCs) towards peri-implant bone tissue, which may be ascribed to the upregulation of local TGF-β1 and PDGF-BB. Besides, the in vitro study revealed that higher Mg ions was beneficial to the improvement of capability in cell adhesion and osteogenic differentiation of BMSCs. Thus, the enhancement in cell migration, cell adhesion and osteogenic differentiation of BMSCs may contribute to an improved tendon graft osteointegration in the Mg group. Our findings in this work may further facilitate clinical applications of Mg-based interference screws for enhancing tendon graft-bone junction healing in patients indicated for ACLR. STATEMENT OF SIGNIFICANCE How to promote tendon-bone junction healing is one of the major challenging issues for satisfactory clinical outcomes in patients after ACL reconstruction. The improvement of bony ingrowth into the tendon graft-bone interface can enhance the tendon graft osteointegration. In this study, we applied Mg based interference screws to fix the tendon graft in rabbits and found the use of Mg screws could accelerate and significantly increase mineralized matrix formation at the tendon-bone interface in animals when compared to those with Ti screws. We elucidated the mechanism behind the favorable effects of Mg screws on the graft healing in both in vitro and in vivo studies from multiscale technologies. The optimized interface structure and function in Mg group may be ascribed to the improved cell migration capability, enhanced cell adhesion strength and promoted osteogenic differentiation ability of BMSCs under the stimuli of Mg ions degraded from implanted Mg screws. Our findings may help us broaden our thinking in the application potential of Mg interference screws in future clinical trials.

Application of ultrasound accelerates the decalcification process of bone matrix without affecting histological and immunohistochemical analysis

Background/Objectives Decalcification of bone specimens is necessary for routine paraffin embedding and sectioning. Ethylenediaminetetraacetic acid (EDTA), a chelating agent for decalcification, maintains bone tissue integrity and histological features but requires long decalcification period, especially for cortical bone with dense mineral matrix. We hypothesised that the application of a newly commercially available ultrasound (US) decalcifier would accelerate decalcification of thick cortical bone specimen in EDTA efficiently and that the working temperature at 30–45°C would not affect histological and immunohistochemical analysis. Comparison was made with traditional decalcification method with regards to quality of tissue morphology and antigenicity. Methods A fresh human cadaveric femoral shaft was sectioned into 5-mm-thick transverse sections. After fixation, the bone slices were divided into two groups: Ultrasound decalcification group (US DeCal), in which bone sections (n = 3) were placed in a US decalcifier (50 W at a frequency of 40kHz) with EDTA solution, and normal decalcification group (Normal DeCal), in which bone sections (n = 3) were decalcified in EDTA without US. The mineral content of the bone sections was measured with micro-computed tomography and dual-energy X-ray absorptiometry at different time points. Rate of calcium extraction was quantified by measuring the calcium concentration in EDTA solution using inductively coupled plasma optical emission spectrometry. After decalcification, the paraffin sections of the decalcified bone were stained with haematoxylin and eosin or immunohistochemical staining of sclerostin. Results Samples in US DeCal contained 2.9 ± 2.8% of the mineral content at Day 6 and were completely decalcified at Day 8. However, sections in Normal DeCal retained 36.3 ± 5.1% and 24.3 ± 4.8% at Day 6 and Day 8, respectively, and took six times longer to complete decalcification. The concentration of calcium in the EDTA solution of the US DeCal group was 70% higher than that of the Normal DeCal group (p < 0.05) in Day 1 and 2. No staining difference was observed in histological sections between the two groups. Conclusion The application of US decalcification significantly shortened the decalcification time in EDTA without causing histological artefacts. The translational potential of this article This article shows that the application of ultrasound in sample decalcification would shorten the duration that decalcification required. This would accelerate the sample processing for routine bone histology in both basic and clinical research and assessments for diagnostic purposes.