Ageing attenuates bone healing by mesenchymal stem cells in a microribbon hydrogel with a murine long bone critical-size defect model.

BACKGROUND: Despite the high incidence of fractures and pseudoarthrosis in the aged population, a potential role for the use of mesenchymal stem cells (MSCs) in the treatment of bone defects in elderly patients has not been elucidated. Inflammation and the innate immune system, including macrophages, play crucial roles in the differentiation and activation of MSCs. We have developed lentivirus-transduced interleukin 4 (IL4) over-expressing MSCs (IL4-MSCs) to polarize macrophages to an M2 phenotype to promote bone healing in an established young murine critical size bone defect model. In the current study, we explore the potential of IL4-MSCs in aged mice. METHODS: A 2 mm femoral diaphyseal bone defect was created and fixed with an external fixation device in 15- to 17-month-old male and female BALB/c mice. Microribbon (µRB) scaffolds (Sc) with or without encapsulation of MSCs were implanted in the defect sites. Accordingly, the mice were divided into three treatment groups: Sc-only, Sc + MSCs, and Sc + IL4-MSCs. Mice were euthanized six weeks after the surgery; subsequently, MicroCT (µCT), histochemical and immunohistochemical analyses were performed. RESULTS: µCT analysis revealed that bone formation was markedly enhanced in the IL4-MSC group. Compared with the Sc-only, the amount of new bone increased in the Sc + MSCs and Sc + IL4-MSC groups. However, no bridging of bone was observed in all groups. H&E staining showed fibrous tissue within the defect in all groups. Alkaline phosphatase (ALP) staining was increased in the Sc + IL4-MSC group. The Sc + IL4-MSCs group showed a decrease in the number of M1 macrophages and an increase in the number of M2 macrophages, with a significant increase in the M2/M1 ratio. DISCUSSION: IL4 promotes macrophage polarization to an M2 phenotype, facilitating osteogenesis and vasculogenesis. The addition of IL4-MSCs in the µRB scaffold polarized macrophages to an M2 phenotype and increased bone formation; however, complete bone bridging was not observed in any specimens. These results suggest that IL4-MSCs are insufficient to heal a critical size bone defect in aged mice, as opposed to younger animals. Additional therapeutic strategies are needed in this challenging clinical scenario.

Gait Variability to Phenotype Common Orthopedic Gait Impairments Using Wearable Sensors.

Mobility impairments are a common symptom of age-related degenerative diseases. Gait features can discriminate those with mobility disorders from healthy individuals, yet phenotyping specific pathologies remains challenging. This study aims to identify if gait parameters derived from two foot-mounted inertial measurement units (IMU) during the 6 min walk test (6MWT) can phenotype mobility impairment from different pathologies (Lumbar spinal stenosis (LSS)-neurogenic diseases, and knee osteoarthritis (KOA)-structural joint disease). Bilateral foot-mounted IMU data during the 6MWT were collected from patients with LSS and KOA and matched healthy controls (N = 30, 10 for each group). Eleven gait parameters representing four domains (pace, rhythm, asymmetry, variability) were derived for each minute of the 6MWT. In the entire 6MWT, gait parameters in all four domains distinguished between controls and both disease groups; however, the disease groups demonstrated no statistical differences, with a trend toward higher stride length variability in the LSS group (p = 0.057). Additional minute-by-minute comparisons identified stride length variability as a statistically significant marker between disease groups during the middle portion of 6WMT (3rd min: p ≤ 0.05; 4th min: p = 0.06). These findings demonstrate that gait variability measures are a potential biomarker to phenotype mobility impairment from different pathologies. Increased gait variability indicates loss of gait rhythmicity, a common feature in neurologic impairment of locomotor control, thus reflecting the underlying mechanism for the gait impairment in LSS. Findings from this work also identify the middle portion of the 6MWT as a potential window to detect subtle gait differences between individuals with different origins of gait impairment.

Resting-state Amplitude of Low-frequency Fluctuation is a Potentially Useful Prognostic Functional Biomarker in Cervical Myelopathy.

BACKGROUND: Cervical MRI is the standard diagnostic imaging technique for patients with cervical myelopathy. However, the utility of conventional cervical MRI as a predictive biomarker for surgical recovery remains unclear, partly because of the limited information obtained from this anatomically small area. Brain resting-state functional MRI (rs-fMRI) may help identify candidate predictive biomarkers. Two analytical methods that assess local spontaneous brain activity are widely used for rs-fMRI: functional connectivity between two brain regions and amplitude of low-frequency fluctuation (ALFF). In our previous analysis of functional connectivity, we discovered that brain functional connectivity may be a predictive biomarker for neurologic recovery in patients with cervical myelopathy; however, the functional connectivity analysis identified a correlation with only one clinical outcome (the 10-second test). To establish a comprehensive prediction measure, we need to explore other brain biomarkers that can predict recovery of other clinical outcomes in patients with cervical myelopathy. QUESTIONS/PURPOSES: We aimed to (1) elucidate preoperative ALFF alterations in patients with cervical myelopathy and how ALFF changes after surgery, with a focus on postoperative normalization and (2) establish a predictive model using preoperative ALFF by investigating the correlation between preoperative ALFF and postoperative clinical recovery in patients with cervical myelopathy. METHODS: Between August 2015 and June 2017, we treated 40 patients with cervical myelopathy. Thirty patients met our prespecified inclusion criteria, all were invited to participate, and 28 patients opted to do so (93%; 14 men and 14 women; mean age: 67 years). The 28 patients and 28 age- and sex-matched controls underwent rs-fMRI (twice for patients with cervical myelopathy: before and 6 months after cervical decompression surgery). We analyzed the same study population that was used in our earlier study investigating functional connectivity. Controls had none of the following abnormalities: neck or arm pain, visual or auditory disorders, cognitive disorder, structural brain disorder, a history of brain surgery, mental and neurologic disorders, and medications for the central nervous system. We performed ALFF comparisons between preoperative patients with cervical myelopathy and controls, analyzed postoperative ALFF changes in patients with cervical myelopathy, and performed a correlation analysis between preoperative ALFF and clinical recovery in these patients. Clinical outcomes in the cervical myelopathy group were assessed using the 10-second test, the Japanese Orthopaedic Association upper-extremity motor (JOA-UEM) score, JOA upper-extremity sensory score (JOA-UES), and Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire for upper-extremity function (JOACMEQ-UEF) score before and 6 months after surgery, which is when we believe these scores generally reach a plateau. A total of 93% of those enrolled (26 of 28 patients) were analyzed both preoperatively and postoperatively; the other two were lost to follow-up. RESULTS: The cervical myelopathy group had an increase in ALFF in the bilateral primary sensorimotor cortices (right, cluster size = 850 voxels, t-value = 6.10; left, cluster size = 370 voxels, t-value = 4.84) and left visual cortex (cluster size = 556 voxels, t-value = 4.21) compared with the control group. The cervical myelopathy group had a decrease in ALFF in the bilateral posterior supramarginal gyrus (right, cluster size = 222 voxels, t-value = 5.09; left, cluster size = 436 voxels, t-value = 5.28). After surgery, the bilateral sensorimotor cortices (right, cluster size = 468 voxels, t-value = 6.74; left, cluster size = 167 voxels, t-value = 5.40) and left visual cortex (cluster size = 3748 voxels, t-value = 6.66) showed decreased ALFF compared with preoperative ALFF, indicating postoperative normalization of spontaneous brain activities in these regions. However, the bilateral posterior supramarginal gyrus did not show an increase in ALFF postoperatively, although ALFF in this region decreased preoperatively. Greater levels of ALFF at the left and right frontal pole and left pars opercularis of the inferior frontal gyrus before surgery in the cervical myelopathy group were correlated with larger improvements in the JOACMEQ-UEF score 6 months after surgery (r = 0.784; p < 0.001, r = 0.734; p < 0.001 and r = 0.770, respectively; p < 0.001). The prediction formula, based on preoperative ALFF values in the left frontal pole, was as follows: the predicted postoperative improvement in the JOACMEQ-UEF score = 34.6 × preoperative ALFF value - 7.0 (r = 0.614; p < 0.001). CONCLUSIONS: Our findings suggest that preoperative ALFF may be a biomarker for postoperative recovery in that it predicted postoperative JOACMEQ-UEF scores. To establish a comprehensive prediction measure for neurologic recovery in patients with cervical myelopathy, a multicenter study is underway. LEVEL OF EVIDENCE: Level II, diagnostic study.

BMP and TGFß use and release in bone regeneration

A fracture that does not unite in nine months is defined as nonunion. Nonunion is common in fragmented fractures and large bone defects where vascularization is impaired. The distal third of the tibia, the scaphoid bone or the talus fractures are furthermore prone to nonunion. Open fractures and spinal fusion cases also need special monitoring for healing. Bone tissue regeneration can be attained by autografts, allografts, xenografts and synthetic materials, however their limited availability and the increased surgical time as well as the donor site morbidity of autograft use, and lower probability of success, increased costs and disease transmission and immunological reaction probability of allografts oblige us to find better solutions and new grafts to overcome the cons. A proper biomaterial for regeneration should be osteoinductive, osteoconductive, biocompatible and mechanically suitable. Cytokine therapy, where growth factors are introduced either exogenously or triggered endogenously, is one of the commonly used method in bone tissue engineering. Transforming growth factor ß (TGFß) superfamily, which can be divided structurally into two groups as bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs) and TGFß, activin, Nodal branch, Mullerian hormone, are known to be produced by osteoblasts and other bone cells and present already in bone matrix abundantly, to take roles in bone homeostasis. BMP family, as the biggest subfamily of TGFß superfamily, is also reported to be the most effective growth factors in bone and development, which makes them one of the most popular cytokines used in bone regeneration. Complications depending on the excess use of growth factors, and pleiotropic functions of BMPs are however the main reasons of why they should be approached with care. In this review, the Smad dependent signaling pathways of TGFß and BMP families and their relations and the applications in preclinical and clinical studies will be briefly summarized.

Difference in the fusion rate and bone formation between artificial bone and iliac autograft inside an inter-body fusion cage - A comparison between porous hydroxyapatite/type 1 collagen composite and autologous iliac bone.

BACKGROUND: Lateral inter-body fusion (LIF) using cages with a large bone grafting space can lead to a shortage of autologous grafting materials. The use of artificial bone is an option to increase the volume of grafting materials. However, the rate of bony fusion for these materials compared to that of autologous bone is unclear. METHODS: The bone fusion rate for artificial bone (HAp/Col) and autologous iliac bone (IBG) graft among 23 patients who had undergone LIF (total 66 disc levels) combined with multilevel posterior corrective fusion for the treatment of adult spinal deformity was retrospectively evaluated. To allow comparison, one of the two separate bone grafting holes in each LIF cage was filled with HAp/Col and the other, with IBG. The change in Hounsfield units (HU) inside the implanted holes at 1-year post surgery (PO1Y) from baseline and immediately after surgery and bony fusion between adjacent vertebrae, defined by the extent of trabecular continuity at PO1Y, were evaluated using computed tomography. Differences between the convex and concave sides as well as effects of the side of approach were investigated. RESULTS: HU values increased significantly for IBG, from 228.9 at baseline to 286.1 at PO1Y (p < 0.001), with no change for HAp/Col. The fusion rate was higher for IBG (71.2%) than for HAp/Col (19.7%; p < 0.001). A significant effect of the location of the holes on fusion rate was identified for HAp/Col but not IBG. No effects of the side of approach were identified. CONCLUSIONS: A higher rate of fusion in LIF cages was obtained with IBG than with HAp/Col, with no effect of location of implantation (convex or concave) for IBG. Therefore, exclusive use of artificial bone, particularly on the convex side, should be avoided during LIF.

Outcomes of modified metatarsal shortening offset osteotomy for forefoot deformity in patients with rheumatoid arthritis: Short to mid-term follow-up.

OBJECTIVES: Advances in drug therapy for rheumatoid arthritis (RA) have been encouraging us to preserve the metatarsopharangeal (MTP) joint in correction of forefoot deformities, and original metatarsal shortening offset osteotomy was recommended as one of the conventional surgical options for forefoot deformities in RA cases. The objective of this study was to evaluate short- to mid-term outcomes of modified metatarsal shortening offset osteotomy. METHODS: A retrospective observational study was completed for 80 RA cases (mean follow-up period: 3.2 years) who underwent modified metatarsal shortening offset osteotomy. Both lesser toe scales and RA foot ankle scales were administered using the Japanese Society for Surgery of the Foot (JSSF) standard rating system, and a postoperative self-administered foot evaluation questionnaire (SAFE-Q) at final follow-up was also checked to evaluate clinical outcomes. RESULTS: This procedure significantly improved clinical scores of both the JSSF [lesser toes and RA foot and ankle] scales. Of 80 feet, 24 (30%) showed recurrence of MTP joint subluxation/dislocation. Furthermore, the feet in the recurrence group showed significant varus hindfoot. On the other hand, valgus foot in the recurrence group more frequently included midfoot bony ankyloses. All of the affected feet showed the limitation of MTP joints (<70°) after surgery. CONCLUSIONS: Modified metatarsal shortening offset osteotomy was recommended for RA forefoot disorders as one of the joint preservation surgeries in short- to mid-term follow-up. However, some modifications to avoid limitation of ROM in the MTP joint are required. It must be borne in mind that varus hindfoot and/or bony ankyloses in the mid-hindfoot can cause recurrence of dorsal dislocation/subluxation of the lesser toe MTP joint.

Sex differences of NF-κB-targeted therapy for mitigating osteoporosis associated with chronic inflammation of bone.

Aims: Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice. Methods: We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR). Results: Local delivery of NF-κB decoy ODN in vivo increased osteogenesis in males, but not females, in the presence of chronic inflammation induced by cPE. Bone resorption activity was decreased in both sexes. In vitro osteogenic and osteoclastic differentiation assays during inflammatory conditions did not reveal differences among the groups. Receptor activator of nuclear factor kappa Β ligand (Rankl) gene expression by osteoblasts was significantly decreased only in males when treated with ODN. Conclusion: We demonstrated that NF-κB decoy ODN increased osteogenesis in male mice and decreased bone resorption activity in both sexes in preclinical models of chronic inflammation. NF-κB signalling could be a therapeutic target for chronic inflammatory diseases involving bone, especially in males.

Preclinical models for studying corticosteroid-induced osteonecrosis of the femoral head.

Nontraumatic osteonecrosis of the femoral head (ONFH) is a refractory condition that commonly results in femoral head collapse and degenerative arthritis of the hip. In the early stages, surgical procedures for hip preservation, including core decompression (CD), have been developed to prevent progressive collapse of the femoral head. Optimization of bone regeneration and biological augmentation may further enhance the therapeutic efficacy of CD for ONFH. Thus, combining CD with cell-based therapy has recently been proposed. In fact, patients treated with cell-based therapy using autologous bone marrow concentrate demonstrate improved survivorship of the femoral head, compared with conventional CD alone. Preclinical research studies to investigate adjunctive therapies for CD often utilize the rabbit model of corticosteroid-induced ONFH. Mesenchymal stem cells (MSCs) are known to promote osteogenesis and angiogenesis, and decrease inflammation in bone. Local drug delivery systems have the potential to achieve targeted therapeutic effects by precisely controlling the drug release rate. Scaffolds can provide an osteoconductive structural framework to facilitate the repair of osteonecrotic bone tissue. We focused on the combination of both cell-based and scaffold-based therapies for bone tissue regeneration in ONFH. We hypothesized that combining CD and osteoconductive scaffolds would provide mechanical strength and structural cell guidance; and that combining CD and genetically modified (GM) MSCs to express relevant cytokines, chemokines, and growth factors would promote bone tissue repair. We developed GM MSCs that overexpress the anti-inflammatory, pro-reconstructive cytokines platelet-derived growth factor-BB to provide MSCs with additional benefits and investigated the efficacy of combinations of these GM MSCs and scaffolds for treatment of ONFH in skeletally mature male New Zealand white rabbits. In the future, the long-term safety, efficacy, durability, and cost-effectiveness of these and other biological and mechanical treatments must be demonstrated for the patients affected by ONFH.

CCL2 promotes osteogenesis by facilitating macrophage migration during acute inflammation.

Novel minimally invasive strategies are needed to obtain robust bone healing in complex fractures and bone defects in the elderly population. Local cell therapy is one potential option for future treatment. Mesenchymal stromal cells (MSCs) are not only involved in osteogenesis but also help direct the recruitment of macrophages during bone regeneration via MSC-macrophage crosstalk. The C-C motif chemokine ligand 2 (CCL2) is an inflammatory chemokine that is associated with the migration of macrophages and MSCs during inflammation. This study investigated the use of CCL2 as a therapeutic target for local cell therapy. MSCs and macrophages were isolated from 10 to 12 week-old BALB/c male mice. Genetically modified CCL2 over-expressing MSCs were produced using murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture. Cell migration assays were also performed. MSCs transfected with murine CCL2-secreting pCDH-CMV-mCCL2-copGFP expressing lentivirus vector showed higher levels of CCL2 secretion compared to unaltered MSCs (p < 0.05). Genetic manipulation did not affect cell proliferation. CCL2 did not affect the osteogenic ability of MSCs alone. However, acute (1 day) but not sustained (7 days) stimulation with CCL2 increased the alizarin red-positive area when MSCs were co-cultured with macrophages (p < 0.001). Both recombinant CCL2 (p < 0.05) and CCL2 released from MSCs (p < 0.05) facilitated macrophage migration. We demonstrated that acute CCL2 stimulation promoted subsequent osteogenesis in co-culture of MSCs and macrophages. Acute CCL2 stimulation potentially facilitates osteogenesis during the acute inflammatory phase of bone healing by directing local macrophage migration, fostering macrophage-MSC crosstalk, and subsequently, by activating or licensing of MSCs by macrophage pro-inflammatory cytokines. The combination of CCL2, MSCs, and macrophages could be a potential strategy for local cell therapy in compromised bone healing.

Bone regeneration in inflammation with aging and cell-based immunomodulatory therapy.

Aging of the global population increases the incidence of osteoporosis and associated fragility fractures, significantly impacting patient quality of life and healthcare costs. The acute inflammatory reaction is essential to initiate healing after injury. However, aging is associated with "inflammaging", referring to the presence of systemic low-level chronic inflammation. Chronic inflammation impairs the initiation of bone regeneration in elderly patients. This review examines current knowledge of the bone regeneration process and potential immunomodulatory therapies to facilitate bone healing in inflammaging.Aged macrophages show increased sensitivity and responsiveness to inflammatory signals. While M1 macrophages are activated during the acute inflammatory response, proper resolution of the inflammatory phase involves repolarizing pro-inflammatory M1 macrophages to an anti-inflammatory M2 phenotype associated with tissue regeneration. In aging, persistent chronic inflammation resulting from the failure of M1 to M2 repolarization leads to increased osteoclast activation and decreased osteoblast formation, thus increasing bone resorption and decreasing bone formation during healing.Inflammaging can impair the ability of stem cells to support bone regeneration and contributes to the decline in bone mass and strength that occurs with aging. Therefore, modulating inflammaging is a promising approach for improving bone health in the aging population. Mesenchymal stem cells (MSCs) possess immunomodulatory properties that may benefit bone regeneration in inflammation. Preconditioning MSCs with pro-inflammatory cytokines affects MSCs' secretory profile and osteogenic ability. MSCs cultured under hypoxic conditions show increased proliferation rates and secretion of growth factors. Resolution of inflammation via local delivery of anti-inflammatory cytokines is also a potential therapy for bone regeneration in inflammaging. Scaffolds containing anti-inflammatory cytokines, unaltered MSCs, and genetically modified MSCs can also have therapeutic potential. MSC exosomes can increase the migration of MSCs to the fracture site and enhance osteogenic differentiation and angiogenesis.In conclusion, inflammaging can impair the proper initiation of bone regeneration in the elderly. Modulating inflammaging is a promising approach for improving compromised bone healing in the aging population.

Glycolytic reprogramming in macrophages and MSCs during inflammation.

Background: Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs). Methods: We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE. Results: Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines. Conclusion: This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

C-C Motif Chemokine Ligand 2 Enhances Macrophage Chemotaxis, Osteogenesis, and Angiogenesis during the Inflammatory Phase of Bone Regeneration.

Local cell therapy has recently gained attention for the treatment of joint diseases and fractures. Mesenchymal stem cells (MSCs) are not only involved in osteogenesis and angiogenesis, but they also have immunomodulatory functions, such as inducing macrophage migration during bone regeneration via macrophage crosstalk. C-C motif chemokine ligand 2 (CCL2), a known inflammatory mediator, is associated with the migration of macrophages during inflammation. This study examined the utility of CCL2 as a therapeutic target for local cell therapy. Using lentiviral vectors for rabbit MSCs, genetically modified CCL2 overexpressing MSCs were generated. Osteogenic differentiation assays were performed using MSCs with or without macrophages in co-culture, and cell migration assays were also performed. Additionally, co-cultures were performed with endothelial cells (ECs), and angiogenesis was evaluated using a tube formation assay. Overexpression of CCL2 did not affect bone formation under monoculture conditions but promoted chemotaxis and osteogenesis when co-cultured with macrophages. Furthermore, CCL2-overexpression promoted tube formation in co-culture with ECs. These results suggest that CCL2 induces macrophage chemotaxis and osteogenesis by promoting crosstalk between MSCs and macrophages; CCL2 also stimulates ECs to induce angiogenesis. These findings indicate that CCL2 may be a useful therapeutic target for local cell therapy in areas of bone loss.

Therapeutic effects of MSCs, genetically modified MSCs, and NFĸB-inhibitor on chronic inflammatory osteolysis in aged mice.

The number of total joint replacements is increasing, especially in elderly patients, and so too are implant-related complications such as prosthesis loosening. Wear particles from the prosthesis induce a chronic inflammatory reaction and subsequent osteolysis, leading to the need for revision surgery. This study investigated the therapeutic effect of NF-ĸB decoy oligodeoxynucleotides (ODN), mesenchymal stem cells (MSCs), and genetically-modified NF-ĸB sensing interleukin-4 over-secreting MSCs (IL4-MSCs) on chronic inflammation in aged mice. The model was generated by continuous infusion of contaminated polyethylene particles into the intramedullary space of the distal femur of aged mice (15-17 months old) for 6 weeks. Local delivery of ODN showed increased bone mineral density (BMD), decreased osteoclast-like cells, increased alkaline phosphatase (ALP)-positive area, and increased M2/M1 macrophage ratio. Local injection of MSCs and IL4-MSCs significantly decreased osteoclast-like cells and increased the M2/M1 ratio, with a greater trend for IL4-MSCs than MSCs. MSCs significantly increased ALP-positive area and BMD values compared with the control. The IL4-MSCs demonstrated higher values for both ALP-positive area and BMD. These findings demonstrated the therapeutic effects of ODN, MSCs, and IL4-MSCs on chronic inflammatory osteolysis in aged mice. The two MSC-based therapies were more effective than ODN in increasing the M2/M1 macrophage ratio, reducing bone resorption, and increasing bone formation. Specifically, MSCs were more effective in increasing bone formation, and IL4-MSCs were more effective in mitigating inflammation. This study suggests potential therapeutic strategies for treating wear particle-associated inflammatory osteolysis after arthroplasty in the elderly.

Metabolic profile of mesenchymal stromal cells and macrophages in the presence of polyethylene particles in a 3D model.

BACKGROUND: Continuous cross talk between MSCs and macrophages is integral to acute and chronic inflammation resulting from contaminated polyethylene particles (cPE); however, the effect of this inflammatory microenvironment on mitochondrial metabolism has not been fully elucidated. We hypothesized that (a) exposure to cPE leads to impaired mitochondrial metabolism and glycolytic reprogramming and (b) macrophages play a key role in this pathway. METHODS: We cultured MSCs with/without uncommitted M0 macrophages, with/without cPE in 3-dimensional gelatin methacrylate (3D GelMA) constructs/scaffolds. We evaluated mitochondrial function (membrane potential and reactive oxygen species-ROS production), metabolic pathways for adenosine triphosphate (ATP) production (glycolysis or oxidative phosphorylation) and response to stress mechanisms. We also studied macrophage polarization toward the pro-inflammatory M1 or the anti-inflammatory M2 phenotype and the osteogenic differentiation of MSCs. RESULTS: Exposure to cPE impaired mitochondrial metabolism of MSCs; addition of M0 macrophages restored healthy mitochondrial function. Macrophages exposed to cPE-induced glycolytic reprogramming, but also initiated a response to this stress to restore mitochondrial biogenesis and homeostatic oxidative phosphorylation. Uncommitted M0 macrophages in coculture with MSC polarized to both M1 and M2 phenotypes. Osteogenesis was comparable among groups after 21 days. CONCLUSION: This work confirmed that cPE exposure triggers impaired mitochondrial metabolism and glycolytic reprogramming in a 3D coculture model of MSCs and macrophages and demonstrated that macrophages cocultured with MSCs undergo metabolic changes to maintain energy production and restore homeostatic metabolism.

The efficiency of genetically modified mesenchymal stromal cells combined with a functionally graded scaffold for bone regeneration in corticosteroid-induced osteonecrosis of the femoral head in rabbits.

Core decompression (CD) with mesenchymal stromal cells (MSCs) is an effective therapy for early-stage osteonecrosis of the femoral head (ONFH). Preconditioning of MSCs, using inflammatory mediators, is widely used in immunology and various cell therapies. We developed a three-dimensional printed functionally graded scaffold (FGS), made of ß-TCP and PCL, for cell delivery at a specific location. The present study examined the efficacy of CD treatments with genetically modified (GM) MSCs over-expressing PDGF-BB (PDGF-MSCs) or GM MSCs co-over-expressing IL-4 and PDGF-BB and preconditioned for three days of exposure to lipopolysaccharide and tumor necrosis factor-alpha (IL-4-PDGF-pMSCs) using the FGS for treating steroid-induced ONFH in rabbits. We compared CD without cell-therapy, with IL-4-PDGF-pMSCs alone, and with FGS loaded with PDGF-MSCs or IL-4-PDGF-pMSCs. For the area inside the CD, the bone volume in the CD alone was higher than in both FGS groups. The IL-4-PDGF-pMSCs alone and FGS + PDGF-MSCs reduced the occurrence of empty lacunae and improved osteoclastogenesis. There was no significant difference in angiogenesis among the four groups. The combined effect of GM MSCs or pMSCs and the FGS was not superior to the effect of each alone. To establish an important adjunctive therapy for CD for early ONFH in the future, it is necessary and essential to develop an FGS that delivers biologics appropriately and provides structural and mechanical support.

Effects of rhBMP-2-loaded hydroxyapatite granules/beta-tricalcium phosphate hydrogel (HA/ß-TCP/hydrogel) composite on a rat model of caudal intervertebral fusion.

The effects and inflammation-related side effects of bone morphogenetic protein (BMP)-2 on posterior lumbar interbody fusion are controversial. One of the potential causes for the inconsistent results is the uncontrolled release of BMP-2 from the collagen carrier. Therefore, BMP delivery systems that support effective bone regeneration while attenuating the side effects are strongly sought for. We developed NOVOSIS putty (NP), a novel composite material of hydroxyapatite (HA), beta-tricalcium phosphate (ß-TCP)/hydrogel, and BMP-2, which can sustainably release BMP-2 over 2 weeks. This study was aimed at comparing the effects and side effects of NP and collagen sponge (CS) containing BMP-2 using a rat caudal intervertebral fusion model. The fusion rates of NP with low and high doses of BMP-2 were significantly higher than those of an iliac bone (IB) graft, but those of CS with low and high doses of BMP-2 were not different from those of the IB graft. Furthermore, the incidences of ectopic bone formation and soft tissue swelling were significantly lower in the NP group than in the CS group. The HA/ß-TCP/hydrogel carrier enabled superior bone induction with low-dose BMP-2 and decreased the incidence of side effects caused by high-dose BMP-2 vis-à-vis the collagen carrier.

Selective Retinoic Acid Receptor γ Antagonist 7C is a Potent Enhancer of BMP-Induced Ectopic Endochondral Bone Formation.

Bone morphogenetic proteins (BMPs) have been clinically applied for induction of bone formation in musculoskeletal disorders such as critical-sized bone defects, nonunions, and spinal fusion surgeries. However, the use of supraphysiological doses of BMP caused adverse events, which were sometimes life-threatening. Therefore, safer treatment strategies for bone regeneration have been sought for decades. Systemic administration of a potent selective antagonist of retinoic acid nuclear receptor gamma (RARγ) (7C) stimulated BMP-induced ectopic bone formation. In this study, we developed 7C-loaded poly lactic nanoparticles (7C-NPs) and examined whether local application of 7C enhances BMP-induced bone regeneration. The collagen sponge discs that absorbed recombinant human (rh) BMP-2 were implanted into the dorsal fascia of young adult mice to induce ectopic bone. The combination of rhBMP-2 and 7C-NP markedly increased the total bone volume and thickness of the bone shell of the ectopic bone in a dose-dependent manner compared to those with rhBMP-2 only. 7C stimulated sulfated proteoglycan production, expression of chondrogenic marker genes, and Sox9 reporter activity in both chondrogenic cells and MSCs. The findings suggest that selective RARγ antagonist 7C or the related compounds potentiate the bone inductive ability of rhBMP-2, as well as support any future research to improve the BMP-2 based bone regeneration procedures in a safe and efficient manner.

Sex differences in the therapeutic effect of unaltered versus NFκB sensing IL-4 over-expressing mesenchymal stromal cells in a murine model of chronic inflammatory bone loss.

Wear particles from joint arthroplasties induce chronic inflammation associated with prolonged upregulation of nuclear factor kappa-B (NF-κB) signaling in macrophages and osteoclasts, which leads to osteolysis and implant loosening. Mesenchymal stromal cell (MSC)-based therapy showed great potential for immunomodulation and mitigation of osteolysis in vivo, especially in the chronic phase of inflammation. We previously generated genetically modified MSCs that secrete the anti-inflammatory cytokine interleukin 4 (IL-4) in response to NF-κB activation (NFκB-IL-4 MSCs). However, whether the impact of sexual difference in the internal environment can alter the therapeutic effects of IL-4 over-secreting MSCs that simultaneously mitigate prolonged inflammation and enhance bone formation remains unknown. This study investigated the therapeutic effects of unaltered MSCs versus NFκB-IL-4 MSCs in mitigating chronic inflammation and enhancing bone formation in male and female mice. The murine model was established by continuous infusion of polyethylene particles contaminated with lipopolysaccharide (cPE) into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation. Unaltered MSCs or NFκB-IL-4 MSCs were infused into the femoral intramedullary cavity in sex-matched groups beginning 3 weeks after primary surgery. Femurs were harvested at 6 weeks, and bone marrow density was measured with micro-computational tomography. Numbers of osteoclast-like cells, osteoblasts, and macrophages were evaluated with histochemical and immunofluorescence staining. cPE infusion resulted in severe bone loss at the surgery site, increased tartrate-resistant acid phosphatase positive osteoclasts and M1 pro-inflammatory macrophages, and decreased alkaline phosphatase expression. MSC-based therapy effectively decreased local bone loss and polarized M1 macrophages into an M2 anti-inflammatory phenotype. In females, unaltered MSCs demonstrated a larger impact in enhancing the osteogenesis, but they demonstrated similar anti-inflammatory effects compared to NFκB-IL-4 MSCs. These results demonstrated that local inflammatory bone loss can be effectively modulated via MSC-based treatments in a sexually dimorphic manner, which could be an efficacious therapeutic strategy for treatment of periprosthetic osteolysis in both genders.

A novel BMP-2-loaded hydroxyapatite/beta-tricalcium phosphate microsphere/hydrogel composite for bone regeneration.

Although bone morphogenetic protein (BMP) has potent osteoinductivity, the potential adverse events attributed to its burst release prevent its widespread clinical application. Therefore, there is a strong need for BMP delivery systems that maximize osteoinductivity while preventing adverse effects. We evaluated the bone-regenerating potential of NOVOSIS putty (NP), a novel composite combining hydroxyapatite, beta-tricalcium phosphate microsphere/poloxamer 407-based hydrogel, and recombinant human (rh) BMP-2. In vitro assessment of release kinetics by enzyme-linked immunosorbent assay demonstrated sustained release of rhBMP-2 from NP and burst release from collagen sponge (CS), and in vivo assessment of release kinetics by longitudinal tracking of fluorescently labeled rhBMP-2 showed a longer biological half-life of rhBMP-2 with NP than with CS. Furthermore, osteogenic gene expression in MC3T3-E1 cells was significantly higher after co-culture with NP than after co-culture with CS, suggesting that the sustained release of rhBMP-2 from NP effectively contributed to the differentiation of osteoblasts. In a rat spinal fusion model, the volume and quality of newly formed bone was higher in the NP group than in the CS group. Use of NP results in efficient bone regeneration through sustained release of rhBMP-2 and improves the quality of BMP-induced bone.

Effect on Osteogenic Differentiation of Genetically Modified IL4 or PDGF-BB Over-Expressing and IL4-PDGF-BB Co-Over-Expressing Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro.

The use of genetically modified (GM) mesenchymal stromal cells (MSCs) and preconditioned MSCs (pMSCs) may provide further opportunities to improve the outcome of core decompression (CD) for the treatment of early-stage osteonecrosis of the femoral head (ONFH). GM interleukin-4 (IL4) over-expressing MSCs (IL4-MSCs), platelet-derived growth factor (PDGF)-BB over-expressing MSCs (PDGF-BB-MSCs), and IL4-PDGF-BB co-over-expressing MSCs (IL4-PDGF-BB-MSCs) and their respective pMSCs were used in this in vitro study and compared with respect to cell proliferation and osteogenic differentiation. IL4-MSCs, PDGF-BB-MSCs, IL4-PDGF-BB-MSCs, and each pMSC treatment significantly increased cell proliferation compared to the MSC group alone. The percentage of Alizarin red-stained area in the IL4-MSC and IL4-pMSC groups was significantly lower than in the MSC group. However, the percentage of Alizarin red-stained area in the PDGF-BB-MSC group was significantly higher than in the MSC and PDGF-BB-pMSC groups. The percentage of Alizarin red-stained area in the IL4-PDGF-BB-pMSC was significantly higher than in the IL4-PDGF-BB-MSC group. There were no significant differences in the percentage of Alizarin red-stained area between the MSC and IL4-PDGF-BB-pMSC groups. The use of PDGF-BB-MSCs or IL4-PDGF-BB-pMSCs increased cell proliferation. Furthermore, PDGF-BB-MSCs promoted osteogenic differentiation. The addition of GM MSCs may provide a useful supplementary cell-based therapy to CD for treatment of ONFH.