Technology Behind Cell Therapy Augmentation of Fracture Healing: Concentrated Bone Marrow Aspirate.

With an aging population, and an anticipated increase in overall fracture incidence, a sound understanding of bone healing and how technology can optimize this process is crucial. Concentrated bone marrow aspirate (cBMA) is a technology that capitalizes on skeletal stem and progenitor cells (SSPCs) to enhance the regenerative capacity of bone. This overview highlights the science behind cBMA, discusses the role of SSPCs in bone homeostasis and fracture repair, and briefly details the clinical evidence supporting the use of cBMA in fracture healing. Despite promising early clinical results, a lack of standardization in harvest and processing techniques, coupled with patient variability, presents challenges in optimizing the use of cBMA. However, cBMA remains an emerging technology that may certainly play a crucial role in the future of fracture healing augmentation.

Non-psychoactive Cannabidiol Prevents Osteoporosis in an Animal Model and Increases Cell Viability, Proliferation, and Osteogenic Gene Expression in Human Skeletal Stem and Progenitor Cells.

Cannabidiol (CBD), the non-psychoactive component of the Cannabis sativa plant, is marketed as a potential therapeutic agent and has been studied for its roles in reducing inflammation and managing neuropathic pain. Some studies have reported that CB1 and CB2 receptor activation can attenuate and reverse bone loss in experimental animal models. Despite this, little is known about the impact of CBD on fracture healing. We investigated the effects of CBD in vitro using human osteoprogenitor cells and in vivo via murine femur fracture and osteoporosis models. In vitro mesenchymal stem cells were treated with increasing concentrations of crystalized pharmaceutical grade CBD or vehicle solution. Cell viability and proliferation were significantly increased in cells treated with CBD compared to vehicle control. Osteocalcin expression was also significantly higher in the CBD-treated human stem cells compared to vehicle control. In vivo the effect of CBD on bone mineral density and fracture healing in mice was examined using a two-phase experimental approach. Fluoxetine was used for pharmacologic induction of osteoporosis and surgical oophorectomy (OVX) was used for hormonal induction of osteoporosis. X-ray and microCT analysis showed that CBD prevented both fluoxetine- and OVX-induced osteoporosis. We found that while OVX resulted in delayed bone healing in control mice, CBD-pretreated mice exhibited normal bone healing. Collectively these in vitro and in vivo findings suggest that CBD exerts cell-specific effects which can be exploited to enhance bone metabolism. These findings also indicate that CBD usage in an osteoporotic population may positively impact bone morphology, warranting further research.

Rosiglitazone induces adipogenesis of both marrow and periosteum derived mesenchymal stem cells during endochondral fracture healing.

BACKGROUND: Type 2 diabetes mellitus (T2DM) afflicts about six percent of the global population, and these patients suffer from a two-fold increased fracture risk. Thiazolidinediones (TZDs), including rosiglitazone, are commonly used medications in T2DM because they have a low incidence of monotherapy failure. It is known that rosiglitazone is associated with secondary osteoporosis, further increasing the fracture risk in an already susceptible population. However, it is not yet understood how rosiglitazone impacts endochondral bone healing after fracture. The aim of this study is to elucidate how rosiglitazone treatment impacts endochondral fracture healing, and how rosiglitazone influences the differentiation of skeletal stem and progenitor cells from the bone marrow and the periosteum. METHODS: An in-vivo mouse femur fracture model was employed to evaluate differences in fracture healing between mice treated with and without rosiglitazone chow. Fracture healing was assessed with histology and micro computed tomography (µCT). In-vitro assays utilized isolated mouse bone marrow stromal cells and periosteal cells to investigate how rosiglitazone impacts the osteogenic capability and adipogenicity of these cells. RESULTS: The in-vivo mouse femur fracture model showed that fracture callus in mice treated with rosiglitazone had significantly more adipose content than those of control mice that did not receive rosiglitazone. In addition, µCT analysis showed that rosiglitazone treated mice had significantly greater bone volume, but overall greater porosity when compared to control mice. In-vitro experimentation showed significantly less osteogenesis and more adipogenesis in bone marrow derived progenitor cells that were cultured in osteogenic media. In addition, rosiglitazone treatment alone caused significant increases in adipogenesis in both bone marrow and periosteum derived cells. CONCLUSION: Rosiglitazone impairs endochondral fracture healing in mice by increasing adipogenesis and decreasing osteogenesis of both bone marrow and periosteum derived skeletal progenitor cells.

Clinical Effect of Selective Serotonin Reuptake Inhibitors (SSRIs) on Fracture Healing.

PURPOSE: Chronic use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression has been linked to an imbalance in bone metabolism leading to osteoporosis. More recently, the use of SSRIs in murine models has been shown to delay bone healing both in vivo and in vitro by decreasing the osteoblastic differentiation and mineralization. The purpose of this study was to evaluate whether or not chronic use of SSRI's in nonunion patients increases their time to union after surgical intervention. METHODS: We retrospectively analyzed 343 patients in a nonunion database to determine which patients were on SSRI medication. Of these patients, 139 could be contacted and of those 102 were not taking SSRIs and 37 were taking SSRIs. Patient's time to union from nonunion surgical intervention between each cohort at our institution was recorded as the primary outcome. Patient's medical comorbidities that could affect union rates such as diabetes and smoking status were also noted. Baseline Short Musculoskeletal Function Assessment (SMFA) index for bother and function were recorded from the time of nonunion surgery as well as last follow-up. RESULTS: Compared to recent census data, we found significantly more patients in the nonunion cohort using SSRIs (26.6%) than patients in the general population using any type of antidepressant (11%). There was no significant difference in the patients' baseline characteristics other than patients on SSRI treatment had a higher body mass index (BMI) and age (p = 0.048 and p = 0.043, respectively). There was no significant difference noted in the fracture types (p = 0.2063). Patients on SSRIs had a higher SMFA bother index and function index on follow-up (p = 0.0103, p = 0.0147). Patients in the SSRI group had a mean time to union from nonunion surgery of 6.1 months compared to 6.0 in patients without SSRI usage (p = 0.74). These did not reach statistical significance when subcohort analysis for long bone fractures was performed for the femur, tibia, and humerus. CONCLUSION: To our knowledge, this is the first clinical study to investigate the effects of SSRIs on fracture healing. While in vivo and in vitro murine models have shown that SSRIs can have a deleterious effect on osteoblastic activity, our retrospective analysis did not show a significant difference in time to union between patients with chronic SSRI use and patients who have not been on SSRIs. However, this investigation did show a higher incidence of SSRI use in the nonunion cohort when compared to the general population. In the context of the recent animal model study, this may point to a negative effect of SSRI use on the acute fracture healing process.

Iliac Crest and Distal Radius Autografts Exhibit Distinct Cell-Intrinsic Functional Differences.

PURPOSE: Autologous bone grafts demonstrate osteoconductive, osteoinductive, and osteogenic properties. Hand surgeons commonly augment surgical fixation with autografts to promote fracture healing. This study compared the intrinsic stem cell-like properties of 2 commonly used autograft sources in hand surgery: the iliac crest and distal radius. METHODS: A total of 9 subjects who received an iliac crest bone graft and distal radius bone graft harvest as a part of the standard care of distal radius malunion or nonunion correction or scaphoid nonunion open reduction and internal fixation were enrolled in the study. Cells were isolated by serial collagenase digestion and subjected to fibroblast colony-forming units, osteogenesis, and adipogenesis assays. The expression levels of genes involved in osteogenesis and adipogenesis were confirmed using quantitative polymerase chain reaction. RESULTS: The cells isolated from the iliac crest bone graft compared with those isolated from the distal radius bone graft demonstrated significantly higher mean fibroblast colony-forming unit efficiency; increased osteogenesis, as measured using alizarin red quantification; increased adipogenesis, as measured using oil red O quantification; and higher expression levels of genes involved in osteogenesis and adipogenesis under the respective differentiation conditions. CONCLUSIONS: The cells isolated from the iliac crest bone graft demonstrated a higher fibroblast colony-forming unit capacity and an increased capability to undergo both osteogenesis and adipogenesis. CLINICAL RELEVANCE: Limited evidence exists comparing the intrinsic stem cell-like properties of the iliac crest and distal radius despite the widespread use of each source in hand and wrist surgery. The information from this investigation may assist hand and wrist surgeons with the selection of a source of autograft.

Free Tissue Transfer with Distraction Osteogenesis and Masquelet Technique Is Effective for Limb Salvage in Patients with Gustilo Type IIIB Open Fractures.

Osteocutaneous reconstruction can be challenging because of concomitant injuries and limited donor sites. There is a paucity of data on limb salvage outcomes following combined soft-tissue reconstruction and bone transport or Masquelet procedures. The authors reviewed a consecutive series of open tibia fracture patients undergoing soft-tissue reconstruction with either distraction osteogenesis or Masquelet technique. Endpoints were perioperative flap complications and bone union. Fourteen patients with Gustilo type IIIB open tibia fractures were included. Half of the group received muscle flaps and the remaining half received fasciocutaneous flaps. Ten patients (71.4 percent) underwent distraction osteogenesis and the remaining patients underwent Masquelet technique. Average bone gap length was 65.7 ± 31.3 mm (range, 20 to 120 mm). In the bone transport group, the average external fixation duration was 245 days (range, 47 to 686 days). In the Masquelet group, the average duration of the first stage of this two-stage procedure (i.e., time from cement spacer placement to bone grafting) was 95 days (range, 42 to 181 days). Bone union rate, as determined by radiographic evidence, was 85.7 percent. There was one complete flap failure (7.1 percent). One patient underwent below-knee amputation after failing bone transport and developing chronic osteomyelitis and subsequent infected nonunion. Our case series demonstrates that nonosteocutaneous flap methods of limb reconstruction are a viable option in patients with segmental long bone defects, with a bone union rate of 85 percent and a limb salvage rate over 90 percent in patients with Gustilo type IIIB fractures. CLINICAL QUESTION/LEVEL OF EVIDENCE:: Therapeutic, IV.

Gammaherpesvirus Infection of Human Neuronal Cells.

UNLABELLED: Gammaherpesviruses human herpesvirus 4 (HHV4) and HHV8 are two prominent members of the herpesvirus family associated with a number of human cancers. HHV4, also known as Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus prevalent in 90 to 95% of the human population, is clinically associated with various neurological diseases such as primary central nervous system lymphoma, multiple sclerosis, Alzheimer's disease, cerebellar ataxia, and encephalitis. However, the possibility that EBV and Kaposi's sarcoma-associated herpesvirus (KSHV) can directly infect neurons has been largely overlooked. This study has, for the first time, characterized EBV infection in neural cell backgrounds by using the Sh-Sy5y neuroblastoma cell line, teratocarcinoma Ntera2 neurons, and primary human fetal neurons. Furthermore, we also demonstrated KSHV infection of neural Sh-Sy5y cells. These neuronal cells were infected with green fluorescent protein-expressing recombinant EBV or KSHV. Microscopy, genetic analysis, immunofluorescence, and Western blot analyses for specific viral antigens supported and validated the infection of these cells by EBV and KSHV and showed that the infection was efficient and productive. Progeny virus produced from infected neuronal cells efficiently infected fresh neuronal cells, as well as peripheral blood mononuclear cells. Furthermore, acyclovir was effective at inhibiting the production of virus from neuronal cells similar to lymphoblastoid cell lines; this suggests active lytic replication in infected neurons in vitro. These studies represent a potentially new in vitro model of EBV- and KSHV-associated neuronal disease development and pathogenesis. IMPORTANCE: To date, no in vitro study has demonstrated gammaherpesvirus infection of neuronal cells. Moreover, worldwide clinical findings have linked EBV to neuronal pathologies, including multiple sclerosis, primary central nervous system lymphoma, and Alzheimer's disease. In this study, for the first time, we have successfully demonstrated the in vitro infection of Sh-Sy5y and Ntera2 cells, as well as human primary neurons. We have also determined that the infection is predominately lytic. Additionally, we also report infection of neuronal cells by KSHV in vitro similar to that by EBV. These findings may open new avenues of consideration related to neuronal pathologies and infection with these viruses. Furthermore, their contribution to chronic infection linked to neuronal disease will provide new clues to potential new therapies.

Kaposi's sarcoma-associated herpesvirus genome programming during the early stages of primary infection of peripheral blood mononuclear cells.

UNLABELLED: The early period of Kaposi's sarcoma-associated herpesvirus (KSHV) infection involves the dynamic expression of viral genes, which are temporally and epigenetically regulated. KSHV can effectively infect and persist in endothelial as well as human B cells with different gene expression patterns. To understand the temporal epigenetic changes which occur when KSHV infects the lymphocytic compartment, we infected human peripheral blood mononuclear cells (PBMCs) and comprehensively analyzed the changes which occurred at the binding sites of virally encoded lytic as well as latent proteins along with epigenetic modifications across the KSHV genome during early primary infection. Using chromatin immunoprecipitation (ChIP) assays, we showed that the KSHV genome acquires a uniquely distinct histone modification pattern of methylation (H3K4me3, H3K9me3, and H3K27me3) and acetylation (H3Ac) during de novo infection of human PBMCs. This pattern showed that the epigenetic changes were temporally controlled. The binding profiles of KSHV latent protein LANA and the immediate early proteins RTA and K8 showed specific patterns at different times postinfection, which reflects the gene expression program. Further analysis demonstrated that KSHV can concurrently express lytic and latent genes which were associated with histone modifications at these specific regions on the viral genome. We identified three KSHV genes, K3, ORF49, and ORF64, which exhibited different profiles of histone modifications during the early stages of PBMC infection. These studies established a distinct pattern of epigenetic modification which correlates with viral gene expression temporally regulated during the first 7 days of PBMC infection and provides clues to the regulatory program required for successful infection by KSHV of human PBMCs. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) has been documented as one of the major contributors to morbidity and mortality in AIDS patients during the AIDS pandemic. During its life cycle, KSHV undergoes latent and lytic replication. Typically, KSHV maintains a stringent preference for latent infection in the infected B cells. However, 1 to 5% of infected cells undergo spontaneous lytic reactivation. KSHV lytic replication and infection of new cells are likely to be critical for maintaining the population of infected cells which drive virus-associated pathogenesis. Here, we explored the temporal changes of crucial histone marks on the KSHV genome during early infection of human primary peripheral blood mononuclear cells (PBMCs), which are a physiologically relevant system for monitoring primary infection. These results showed that KSHV possessed a distinct pattern of epigenetic marks during early infection of PBMCs. Further, KSHV concurrently expressed lytic and latent genes during this early period. These results now provide new evidence which contributes to understanding the molecular mechanism that regulates viral gene expression during early infection.