Methodology and Characterization of a 3D Bone Organoid Model Derived from Murine Cells.

Here, we report on the development of a cost-effective, well-characterized three-dimensional (3D) model of bone homeostasis derived from commonly available stocks of immortalized murine cell lines and laboratory reagents. This 3D murine-cell-derived bone organoid model (3D-mcBOM) is adaptable to a range of contexts and can be used in conjunction with surrogates of osteoblast and osteoclast function to study cellular and molecular mechanisms that affect bone homeostasis in vitro or to augment in vivo models of physiology or disease. The 3D-mcBOM was established using a pre-osteoblast murine cell line, which was seeded into a hydrogel extracellular matrix (ECM) and differentiated into functional osteoblasts (OBs). The OBs mineralized the hydrogel ECM, leading to the deposition and consolidation of hydroxyapatite into bone-like organoids. Fourier-transform infrared (FTIR) spectroscopy confirmed that the mineralized matrix formed in the 3D-mcBOM was bone. The histological staining of 3D-mcBOM samples indicated a consistent rate of ECM mineralization. Type I collagen C-telopeptide (CTX1) analysis was used to evaluate the dynamics of OC differentiation and activity. Reliable 3D models of bone formation and homeostasis align with current ethical trends to reduce the use of animal models. This functional model of bone homeostasis provides a cost-effective model system using immortalized cell lines and easily procured supplemental compounds, which can be assessed by measuring surrogates of OB and OC function to study the effects of various stimuli in future experimental evaluations of bone homeostasis.

A functional three-dimensional microphysiological human model of myeloma bone disease.

Human myeloma bone disease (MBD) occurs when malignant plasma cells migrate to the bone marrow and commence inimical interactions with stromal cells, disrupting the skeletal remodeling process. The myeloma cells simultaneously suppress osteoblastic bone formation while promoting excessive osteoclastic resorption. This bone metabolism imbalance produces osteolytic lesions that cause chronic bone pain and reduce trabecular and cortical bone structural integrity, and often culminate in pathological fractures. Few bone models exist that enable scientists to study MBD and the effect therapies have on restoring the bone metabolism imbalance. The purpose of this research was to develop a well characterized three-dimensional (3D) bone organoid that could be used to study MBD and current or potential treatment options. First, bone marrow stromal cell-derived osteoblasts (OBs) mineralized an endosteal-like extracellular matrix (ECM) over 21 days. Multiple analyses confirmed the generation of hydroxyapatite (HA)-rich bone-like tissue fragments that were abundant in alkaline phosphatase, calcium, and markers of osteoblastic gene expression. On day 22, bone marrow macrophage (BMM)-derived osteoclasts (OCs) were introduced to enhance the resorptive capability of the model and recapitulate the balanced homeostatic nature of skeletal remodeling. Tartrate-resistant acid phosphatase 5b (TRAcP-5b), type I collagen C-telopeptide (CTX-1), and gene expression analysis confirmed OC activity in the normal 3D organoid (3D in vitro model of normal bonelike fragments [3D-NBF]). On day 30, a human multiple myeloma (MM)-derived plasmacytoma cell line was introduced to the 3D-NBF to generate the 3D-myeloma bone disease organoid (3D-MBD). After 12 days, the 3D-MBD had significantly reduced total HA, increased TRAcP-5b levels, increases levels of CTX-1, and decreased expression of osteoblastic genes. Therapeutic intervention with pharmaceutical agents including an immunomodulatory drug, a bisphosphonate, and monoclonal restored HA content and reduced free CTX-1 in a dose-dependent manner. This osteogenically functional model of MBD provides a novel tool to study biological mechanisms guiding the disease and to screen potential therapeutics. © 2021 American Society for Bone and Mineral Research (ASBMR).

Local insulin application has a dose-dependent effect on lumbar fusion in a rabbit model.

The purpose of this study was to determine if locally applied insulin has a dose-responsive effect on posterolateral lumbar fusion. Adult male New Zealand White rabbits underwent posterolateral intertransverse spinal fusions (PLFs) at L5-L6 using suboptimal amounts of autograft. Fusion sites were treated with collagen sponge soaked in saline (control, n = 11), or with insulin at low (5 or 10 units, n = 13), mid (20 units, n = 11), and high (40 units, n = 11) doses. Rabbits were euthanized at 6 weeks. The L5-L6 spine segment underwent manual palpation and radiographic evaluation performed by two fellowship trained spine surgeons blinded to treatment. Differences between groups were evaluated by analysis of variance on ranks followed by post-hoc Dunn's tests. Forty-three rabbits were euthanized at the planned 6 weeks endpoint, while three died or were euthanized prior to the endpoint. Radiographic evaluation found bilateral solid fusion in 10%, 31%, 60%, and 60% of the rabbits from the control and low, mid, and high-dose insulin-treated groups, respectively (p < 0.05). As per manual palpation, 7 of 10 rabbits in the mid-dose insulin group were fused as compared to 1 of 10 rabbits in the control group (p < 0.05). This study demonstrates that insulin enhanced the effectiveness of autograft to increase fusion success in the rabbit PLF model. The study indicates that insulin or insulin-mimetic compounds can be used to promote bone regeneration.

Zinc as a Therapeutic Agent in Bone Regeneration.

Zinc is an essential mineral that is required for normal skeletal growth and bone homeostasis. Furthermore, zinc appears to be able to promote bone regeneration. However, the cellular and molecular pathways through which zinc promotes bone growth, homeostasis, and regeneration are poorly understood. Zinc can positively affect chondrocyte and osteoblast functions, while inhibiting osteoclast activity, consistent with a beneficial role for zinc in bone homeostasis and regeneration. Based on the effects of zinc on skeletal cell populations and the role of zinc in skeletal growth, therapeutic approaches using zinc to improve bone regeneration are being developed. This review focuses on the role of zinc in bone growth, homeostasis, and regeneration while providing an overview of the existing studies that use zinc as a bone regeneration therapeutic.

A Meta-Analysis of Non-Osteoarthritis and Osteoarthritis Chondrocyte Gene Expression to Determine the Efficacy of Autologous Chondrocyte Transplantation as a Viable Treatment Option.

BACKGROUND: Osteoarthritis (OA) is a clinical syndrome characterized by joint failure that is accompanied by pain and functional limitations. OA is the leading cause of chronic disability in elderly and it is estimated that the United States spends $185 billion in management of OA annually. Although OA patients receive both pharmacologic and non-pharmacologic treatments, none of them provide long-lasting treatments. Since 1980s, autologous chondrocyte transplantation (ACT) has been used to regenerate cartilage within focal cartilage defects of young patients without pre-existing OA with increased functionality by 74% to 90%. In this technique, chondrocytes are removed from patients, multiplied in vitro, then implanted into the focal cartilage defect. Our review aimed to compare chondrocyte gene expression profiles of non-OA patients with OA patients to determine if OA-derived chondrocytes could be used for the ACT. METHODS: An extensive literature search was conducted with following criteria:(1) comparing chondrocyte gene expression profiles of OA joint and non-OA joint, or (2)relating to ACT. Ingenuity Pathway Analysis (IPA) was then utilized to analyze the differential chondrocyte gene expression profiles of OA to non-OA patients to identify key associated biological pathways. RESULTS: Differential gene expression profiles were similar between non-OA and OA chondrocytes: including ACAN, COL2A1, COL1A1, SOX 6 (p<0.001-0.05); FN1, COL11A1, MMP7, DLX5, SOX9, MMP2, TGFB1, THBS3, COMP, CILP2, ASPN, IGF2, DPT (p<0.001-0.05), and ADAMTS5, LAMA4 (p<0.01-0.05). CONCLUSION: These genes are important to cartilage function. Therefore, our results suggest that OA-derived chondrocytes may be useful to heal focal cartilage defects using ACT.

The Biology of Bone and Ligament Healing.

This review describes the normal healing process for bone, ligaments, and tendons, including primary and secondary healing as well as bone-to-bone fusion. It depicts the important mediators and cell types involved in the inflammatory, reparative, and remodeling stages of each healing process. It also describes the main challenges for clinicians when trying to repair bone, ligaments, and tendons with a specific emphasis on Charcot neuropathy, fifth metatarsal fractures, arthrodesis, and tendon sheath and adhesions. Current treatment options and research areas are also reviewed.

Method for measuring lipid mediators, proteins, and messenger RNAs from a single tissue specimen.

This article describes a new method for extracting RNA, protein, and lipid mediators from a single tissue specimen. Specifically, mouse bone fracture callus specimens were extracted into a single solution that was processed using three different procedures to measure messenger RNA (mRNA) levels by reverse transcription-quantitative polymerase chain reaction (RTqPCR), cytokines and growth factors using an xMAP method, and lipid mediators by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method has several advantages because it decreases the number of animals necessary for experimentation, allows division of the sample from a homogeneous mixture that reduces sample variability, and uses a solution that protects the integrity of the macromolecules during storage.

Effects of local insulin delivery on subperiosteal angiogenesis and mineralized tissue formation during fracture healing.

Local insulin delivery has been shown to improve osseous healing in diabetic animals. The purpose of this study was to quantify the effects of local intramedullary delivery of saline or Ultralente insulin (UL) on various fracture healing parameters using an in vivo non-diabetic BB Wistar rat model. Quantitation of local insulin levels showed a rapid release of insulin from the fractured femora, demonstrating complete release at 2 days. RT-PCR analysis revealed that the expression of early osteogenic markers (Col1α2, osteopontin) was significantly enhanced with UL treatment when compared with saline controls (p < 0.05). Significant differences in VEGF + cells and vascularity were evident between the treatment and control groups at day 7 (p < 0.05). At day 21, histomorphometric analysis demonstrated a significant increase in percent mineralized tissue in the UL-treated animals compared with controls (p < 0.05), particularly within the subperiosteal region of the fracture callus. Mechanical testing at 4 weeks showed significantly greater mechanical strength for UL-treated animals (p < 0.05), but healing in control animals caught up at 6 weeks post-fracture. These results suggest that the primary osteogenic effect of UL during the early stages of fracture healing (1-3 weeks) is through an increase in osteogenic gene expression, subperiosteal angiogenesis, and mineralized tissue formation.

Osteogenic activity of locally applied small molecule drugs in a rat femur defect model.

The long-term success of arthroplastic joints is dependent on the stabilization of the implant within the skeletal site. Movement of the arthroplastic implant within the bone can stimulate osteolysis, and therefore methods which promote rigid fixation or bone growth are expected to enhance implant stability and the long-term success of joint arthroplasty. In the present study, we used a simple bilateral bone defect model to analyze the osteogenic activity of three small-molecule drug implants via microcomputerized tomography (micro-CT) and histomorphometry. In this study, we show that local delivery of alendronate, but not lovastatin or omeprazole, led to significant new bone formation at the defect site. Since alendronate impedes osteoclast-development, it is theorized that alendronate treatment results in a net increase in bone formation by preventing osteoclast mediated remodeling of the newly formed bone and upregulating osteoblasts.

Effects of nonsteroidal anti-inflammatory drugs on flexor tendon adhesion.

PURPOSE: Besides its anti-inflammatory effects, nonsteroidal anti-inflammatory drug therapy may affect tendon healing and the development of peritendinous adhesions. The purpose of this study was to compare the effect of nonselective (ibuprofen) and COX-2 selective (rofecoxib) nonsteroidal anti-inflammatory drugs on the adhesion formation after tendon repair. METHODS: We assigned 67 rabbits to one of 3 (placebo, ibuprofen, or rofecoxib) groups. The deep flexor tendon was transected, followed by a primary repair. Dosing of the medication began the day after surgery and continued for 27 days. The animals were immobilized in a cast for the first 14 days. Postoperatively, tendon adhesion formation was assessed histologically by calculating the total adhesion in serial axial tendon sections at 3 and 6 weeks and by range of motion measurements at 6 and 12 weeks. We measured range of motion by fixing the metacarpal, applying increasing weight to the free end of the flexor digitorum profundus, and measuring the flexion angle between the metacarpal and the proximal phalanx. Comparison was performed between the treatment groups, as well as to the unoperated forepaws. RESULTS: Based on histology, we found no difference between the treatment groups when determining the percentage of adhesion between the flexor tendon and its sheath. Control unoperated forepaws had a significantly greater range of metacarpophalangeal joint flexion than the surgically repaired groups. At 12 weeks, range of motion in the ibuprofen group was significantly better than the placebo (p=.009) and rofecoxib (p=.009) groups. CONCLUSIONS: Ibuprofen has a more important effect in limiting adhesion formation compared with rofecoxib after flexor tendon repair. Because ibuprofen inhibits both COX-1 and COX-2, whereas rofecoxib only inhibits COX-2, ibuprofen therapy appears to offer a greater beneficial effect on tendon repair by reducing formation of adhesions.

A comparison of the effects of ibuprofen and rofecoxib on rabbit fibula osteotomy healing.

BACKGROUND AND PURPOSE: Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) activity, which is the rate-limiting enzyme in the synthesis of prostaglandins. Previous studies have indicated that NSAID therapy, and in particular NSAIDs that specifically target the inflammatory cyclooxygenase (COX-2), impair bone healing. We compared the effects of ibuprofen and rofecoxib on fibula osteotomy healing in rabbits to determine whether nominal, continuous inhibition of COX-2 with rofecoxib would differentially affect fracture healing more than cyclical inhibition of COX-2 using ibuprofen, which inhibits COX-1 and COX-2 and has a short half-life in vivo. METHODS: Bilateral fibula osteotomies were done in 67 skeletally mature male New Zealand white rabbits. The rabbits were treated with placebo, rofecoxib (12.5 mg once a day), or ibuprofen (50 mg 3 times a day) for 28 days after surgery. Plasma ibuprofen levels were measured by HPLC analysis. Bone healing was assessed by histomorphometry at 3 and 6 weeks after osteotomy, and at 6 and 12 weeks by torsional mechanical testing. RESULTS: Plasma ibuprofen levels peaked and declined between successive doses. Fracture callus morphology was abnormal in the rofecoxib-treated rabbits and torsional mechanical testing showed that fracture healing was impaired. Ibuprofen treatment caused persistence of cartilage within the fracture callus and reduced peak torque at 6 weeks after osteotomy as compared to the fibulas from the placebo-treated rabbits. In the specimens allowed to progress to possible healing, non-union was seen in 5 of the 26 fibulas from the rofecoxib-treated animals as compared to 1 of 24 in the placebo group and 1 of 30 in the ibuprofen treatment group. INTERPRETATION: Continuous COX-2 inhibition as modeled by rofecoxib treatment appears to be more deleterious to fracture repair than cyclical cyclooxygenase inhibition as modeled by ibuprofen treatment. Ibuprofen treatment appeared to delay bone healing based upon the persistence of cartilage within the fracture callus and diminished shear modulus. Despite the ibuprofen-induced delay, rofecoxib treatment produced worse fracture (osteotomy) healing than ibuprofen treatment.

Pharmacological inhibition of 5-lipoxygenase accelerates and enhances fracture-healing.

BACKGROUND: Loss of cyclooxygenase-2 activity is known to impair fracture-healing in animal models and to inhibit heterotopic ossification in humans. Cyclooxygenase-2 is the rate-limiting enzyme involved in the conversion of arachidonic acid into prostaglandins. Arachidonic acid also is a substrate for 5-lipoxygenase, which catalyzes the initial steps in leukotriene synthesis. In contrast to cyclooxygenase-2, genetic ablation of 5-lipoxygenase accelerates and enhances fracture-healing in mice. The goal of this study was to determine if systemic inhibition of 5-lipoxygenase with an orally delivered drug could accelerate fracture-healing. METHODS: Closed femoral fractures were made in Sprague-Dawley rats. The rats were treated with oral doses of vehicle (ninety-five rats), celecoxib (fifty-nine rats), or AA-861 (a 5-lipoxygenase inhibitor; eighty-nine rats). Fracture-healing was measured with use of radiographs, histomorphometry, and biomechanical testing. Effects of drug treatments on callus cell proliferation and gene expression were determined by incorporation of bromodeoxyuridine and quantitative polymerase chain reactions, respectively. RESULTS: AA-861 treatment decreased fracture-bridging time, significantly increased early callus cartilage (5.6-fold; p < 0.001) and bone formation (4.2-fold; p = 0.015), and significantly increased callus mechanical properties compared with the vehicle-treated rat fractures. Callus cell proliferation rate was increased by AA-861 treatment, compared with vehicle, at day 2 after fracture (3.68% compared with 2.08%; p < 0.001; 95% confidence interval, -2.81 to -0.039) but was reduced by celecoxib treatment at day 4 after fracture (4.22% compared with 1.84%; p < 0.001; 95% confidence interval, 2.27 to 4.07). At day 10 after fracture, AA-861 and celecoxib treatment increased Type-II collagen mRNA levels (16.0-fold and 6.1-fold, respectively; p < 0.001 for both), but only AA-861 treatment caused an increase in Type-X collagen mRNA (6.3-fold; p < 0.001). AA-861 treatment significantly increased cyclooxygenase-2 (4.0-fold at day 10; p < 0.001) and osteopontin mRNA levels (3.6-fold at day 7; p = 0.024), while decreasing 5-lipoxygenase mRNA levels (5.6-fold at day 4; p < 0.001). CONCLUSIONS: Systemic inhibition of 5-lipoxygenase with an orally delivered drug significantly accelerated and enhanced fracture-healing in this rat model. Gene expression analysis indicates that cyclooxygenase-2 is necessary for callus chondrocytes to progress into hypertrophy so as to complete endochondral ossification. Conversely, inhibition of 5-lipoxygenase alters the inflammatory response, which enhances callus chondrocyte hypertrophy and accelerates endochondral ossification.

Analgesic effects of p38 kinase inhibitor treatment on bone fracture healing.

Traditional and COX-2 selective non-steroidal anti-inflammatory drug (NSAID) treatment inhibits fracture healing in animal models. This indicates that either the inflammatory phase following a bone fracture is necessary for efficient or sufficient bone regeneration to heal the fracture or COX-2 may have a specific function during bone regeneration unrelated to inflammation. These observations also indicate that NSAID use during fracture healing may be contra-indicated. Thus, identification of different analgesics for fracture pain or other orthopaedic surgical procedures would be of significant clinical benefit. Inhibitors of p38 kinase also have significant analgesic properties. However, p38 kinase is a critical regulator of inflammation. To assess the potential use of p38 kinase inhibition as a therapeutic strategy to manage fracture pain, the analgesic properties of SCIO-469, a p38alpha kinase inhibitor, were assessed in a rat fracture model and compared to other common analgesics. In addition, the effects of SCIO-469 treatment on ultimate fracture healing outcomes were measured by radiography and torsional mechanical testing. The data indicate that SCIO-469 was an effective analgesic. No adverse events related to fracture healing were observed in rats treated with SCIO-469. Immunohistochemistry showed that p38 kinase is activated primarily in the first days following a fracture. These observations suggest that p38alpha kinase inhibition may be an effective therapeutic strategy to manage orthopaedic-related pain. These observations also indicate that COX-2 has a specific function during bone regeneration other than promoting inflammation.