Autologous Bone Marrow Stromal Cells Research Articles

Decellularized laser micro-patterned osteochondral implants exhibit zonal recellularization and self-fixing for osteochondral regeneration in a goat model

BackgroundOsteochondral regeneration has long been recognized as a complex and challenging project in the field of tissue engineering. In particular, reconstructing the osteochondral interface is crucial for determining the effectiveness of the repair. Although several artificial layered or gradient scaffolds have been developed recently to simulate the natural interface, the functions of this unique structure have still not been fully replicated. In this paper, we utilized laser micro-patterning technology (LMPT) to modify the natural osteochondral “plugs” for use as grafts and aimed to directly apply the functional interface unit to repair osteochondral defects in a goat model. MethodsFor in vitro evaluations, the optimal combination of LMPT parameters was confirmed through mechanical testing, finite element analysis, and comparing decellularization efficiency. The structural and biological properties of the laser micro-patterned osteochondral implants (LMP-OI) were verified by measuring the permeability of the interface and assessing the recellularization processes. In the goat model for osteochondral regeneration, a conical frustum-shaped defect was specifically created in the weight-bearing area of femoral condyles using a customized trephine with a variable diameter. This unreported defect shape enabled the implant to properly self-fix as expected. ResultsThe micro-patterning with the suitable pore density and morphology increased the permeability of the LMP-OIs, accelerated decellularization, maintained mechanical stability, and provided two relative independent microenvironments for subsequent recellularization. The LMP-OIs with goat's autologous bone marrow stromal cells in the cartilage layer have securely integrated into the osteochondral defects. At 6 and 12 months after implantation, both imaging and histological assessments showed a significant improvement in the healing of the cartilage and subchondral bone. ConclusionWith the natural interface unit and zonal recellularization, the LMP-OI is an ideal scaffold to repair osteochondral defects especially in large animals. The translational potential of this articleThese findings suggest that such a modified xenogeneic osteochondral implant could potentially be explored in clinical translation for treatment of osteochondral injuries. Furthermore, trimming a conical frustum shape to the defect region, especially for large-sized defects, may be an effective way to achieve self-fixing for the implant.

Intracerebral transplantation of MRI-trackable autologous bone marrow stromal cells for patients with subacute ischemic stroke

Ischemic stroke is one of the leading causes of death and neurological disability worldwide, and stem cell therapy is highly expected to reverse the sequelae. This phase 1/2, first-in-human study evaluated the safety, feasibility, and monitoring of an intracerebral-transplanted magnetic resonance imaging (MRI)-trackable autologous bone marrow stromal cell (HUNS001-01) for patients with subacute ischemic stroke. The study included adults with severe disability due to ischemic stroke. HUNS001-01 cultured with human platelet lysates and labeled with superparamagnetic iron oxide was stereotactically transplanted into the peri-infarct area 47-64days after ischemic stroke onset (dose: 2 or 5×107 cells). Neurological and radiographic evaluations were performed throughout 1 year after cell transplantation. The trial was registered at UMIN Clinical Trial Registry (number UMIN000026130). All seven patients who met the inclusion criteria successfully achieved cell expansion, underwent intracerebral transplantation, and completed 1 year of follow-up. No product-related adverse events were observed. The median National Institutes of Health Stroke Scale and modified Rankin scale scores before transplantation were 13 and 4, which showed improvements of 1-8 and 0-2, respectively. Cell tracking proved that the engrafted cells migrated toward the infarction border area 1-6months after transplantation, and the quantitative susceptibility mapping revealed that cell signals at the migrated area constantly increased throughout the follow-up period up to 34% of that of the initial transplanted site. Intracerebral transplantation of HUNS001-01 was safe and well tolerated. Cell tracking shed light on the therapeutic mechanisms of intracerebral transplantation. This work was supported by the Japan Agency for Medical Research and Development (AMED; JP17bk0104045 and JP20bk0104011).

The Construction of Foot and Ankle Joint by Autologous Oxygen-Releasing Nano-Bionic Scaffold Combined with Bone Marrow Mesenchymal Stem Cells

This study assesses the role of a scaffold constructed by co-cultivating autologous oxygen-releasing nano-bionic materials and bone marrow stromal cells (BMSCs) in joint repairing. A scaffold constructed of autologous oxygen-releasing nano-bionic materials and BMSCs was transplanted into SD rats. The immunofluorescence detected the expression of nuclear antigen (PCNA) and analyzed the proliferation of BMSCs. Reverse transcription polymerase chain reaction (RT-PCR) examined the expression of osteogenic markers and TUNEL staining analyzed BMSCs apoptosis. There was a significant difference in the apoptosis and proliferation cell number of BMSCs in ankle joint between solely autologous oxygen-releasing nano-bionic material scaffold group and composite BMSCs nano-bionic scaffold (P <0.05). There was a statistical difference in PCNA and TUNEL expression between two groups at 4 weeks and 8 weeks (P <0.05). The expression of osteogenesis markers in ankle joint at 1 week, 4 weeks, and 8 weeks were continuously reduced, and the composite of autologous oxygen-releasing nanomaterials and BMSCs increased the expression of osteogenic markers (P <0.05). The composite scaffold constructed by autologous oxygen-releasing nano-bionic materials and BMSCs has a good two-way immune regulation function and is able to carry lipids, proteins, nutritional factors, and growth factors, which can effectively promote tissue engineering repairing and delay the scaffold degradation. Combined with the nano-release system to repair bone tissue, composite material can effectively promote the proliferation of joint osteoblasts and osteogenic differentiation, thus help repairing the ankle joint.

Comparison of compositional MRI techniques to quantify the regenerative potential of articular cartilage: a preclinical minipig model after osteochondral defect treatments with autologous mesenchymal stromal cells and unseeded scaffolds.

The field of orthopedics seeks effective, safer methods for evaluating articular cartilage regeneration. Despite various treatment innovations, non-invasive, contrast-free full quantitative assessments of hyaline articular cartilage's regenerative potential using compositional magnetic resonance (MR) sequences remain challenging. In this context, our aim was to investigate the effectiveness of different MR sequences for quantitative assessment of cartilage and to compare them with the current gold standard delayed gadolinium-enhanced MR imaging of cartilage (dGEMRIC) measurements. We employed ex vivo imaging in a preclinical minipig model to assess knee cartilage regeneration. Standardized osteochondral defects were drilled in the proximal femur of the specimens (n=14), which were divided into four groups. Porcine collagen scaffolds seeded with autologous adipose-derived stromal cells (ASC), autologous bone marrow stromal cells (BMSC), and unseeded scaffolds (US) were implanted in femoral defects. Furthermore, there was a defect group which received no treatment. After 6 months, the specimens were examined using different compositional MR methods, including the gold standard dGEMRIC as well as T1, T2, T2*, and T1ρ techniques. The statistical evaluation involved comparing the defect region with the uninjured tibia and femur cartilage layers and all measurements were performed on a clinical 3T MR Scanner. In the untreated defect group, we observed significant differences in the defect region, with dGEMRIC values significantly lower (404.86±64.2 ms, P=0.018) and T2 times significantly higher (44.24±2.75 ms, P<0.001). Contrastingly, in all three treatment groups (ASC, BMSC, US), there were no significant differences among the three regions in the dGEMRIC sequence, suggesting successful cartilage regeneration. However, T1, T2*, and T1ρ sequences failed to detect such differences, highlighting their lower sensitivity for cartilage regeneration. As expected, dGEMRIC is well suited for monitoring cartilage regeneration. Interestingly, T2 imaging also proved to be a reliable cartilage imaging technique and thus offers a contrast agent-free alternative to the former gold standard for subsequent in vivo studies investigating the cartilage regeneration potential of different treatment modalities.

The Effect of Belantamab Mafodotin on Primary Myeloma–Stroma Co-Cultures: Asymmetrical Mitochondrial Transfer between Myeloma Cells and Autologous Bone Marrow Stromal Cells

Belantamab mafodotin (belamaf) is an afucosylated monoclonal antibody conjugated to the microtubule disrupter monomethyl auristatin-F (MMAF) that targets B cell maturation antigen (BCMA) on the surface of malignant plasma cells. Belamaf can eliminate myeloma cells (MMs) through several mechanisms. On the one hand, in addition to inhibiting BCMA-receptor signaling and cell survival, intracellularly released MMAF disrupts tubulin polymerization and causes cell cycle arrest. On the other hand, belamaf induces effector cell-mediated tumor cell lysis via antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In our in vitro co-culture model, the consequences of the first mentioned mechanism can be investigated: belamaf binds to BCMA, reduces the proliferation and survival of MMs, and then enters the lysosomes of malignant cells, where MMAF is released. The MMAF payload causes a cell cycle arrest at the DNA damage checkpoint between the G2 and M phases, resulting in caspase-3-dependent apoptosis. Here, we show that primary MMs isolated from different patients can vary widely in terms of BCMA expression level, and inadequate expression is associated with extremely high resistance to belamaf according to our cytotoxicity assay. We also reveal that primary MMs respond to increasing concentrations of belamaf by enhancing the incorporation of mitochondria from autologous bone marrow stromal cells (BM-MSCs), and as a consequence, MMs become more resistant to belamaf in this way, which is similar to other medications we have analyzed previously in this regard, such as proteasome inhibitor carfilzomib or the BCL-2 inhibitor venetoclax. The remarkable resistance against belamaf observed in the case of certain primary myeloma cell cultures is a cause for concern and points towards the use of combination therapies to overcome the risk of antigen escape.

Effect of combined intervention of exercise and autologous bone marrow stromal cell transplantation on neurotrophic factors and pain-related cascades over time after sciatic nerve injury.

The purpose of this study was to determine whether combined inter-vention of treadmill exercise and bone marrow stromal cell (BMSC) transplantation would affect the expression of neurotrophic factors in the sciatic nerve injury (SNI) and neuropathic pain-related cascades in ipsilateral lumbar 4-5 dorsal root ganglion (DRG) during the early or late stage of sciatic nerve regeneration. The rats were randomly divided into the normal control group (CONT, n=6), sedentary group (SS, n=24), exercise group (SE, n=24), BMSC transplantation group (SB, n=24), BMSC transplantation+exercise group (SBE, n=24) 1, 2, 3, and 5 weeks after SNI. Single dose of 5×106 harvested BMSC was injected into the injury area sing by a 30 gauge needle. Treadmill exercise was performed at a speed of 8 m/min for 30 min once a day. Tropomyosin-receptor kinase B, brain-derived neurotrophic factor and ciliary neurotrophic fac-tor were significantly upregulated in the SE and SBE groups at 1- and 2-week postinjury than those in the CONT and SS groups, and SB and SBE groups continuously kept up proinflammatory cytokines until the late stage of regeneration. Nuclear factor kappa-light-chain-enhancer of activated B cells, interleukin and tumor necrosis factor alpha in ipsi-lateral DRG were progressively decreased by exercise alone application and/or BMSC transplantation at early and late stage of regeneration. Present results provide reliable information that combined intervention of treadmill exercise and BMSC transplantation might be one of the effective treatment strategies for recovering sciatic nerve injury-induced neuropathic pain over time.

A Clinical Study of Alveolar Bone Tissue Engineering Using Autologous Bone Marrow Stromal Cells: Effect of Optimized Cell-Processing Protocol on Efficacy.

(1) Objectives: The effect of cell-processing protocols on the clinical efficacy of bone tissue engineering is not well-known. To maximize efficacy, we optimized the cell-processing protocol for bone-marrow-derived mesenchymal stromal cells for bone tissue engineering. In this study, the efficacy of bone tissue engineering using this modified protocol was compared to that of the original protocol. (2) Materials and Methods: This single-arm clinical study included 15 patients. Cells were obtained from bone marrow aspirates and expanded in culture flasks containing basic fibroblast growth factor. The cells were seeded onto β-tricalcium phosphate granules and induced into osteogenic cells for two weeks. Then, the cell-scaffold composites were transplanted into patients with severe atrophic alveolar bone. Radiographic evaluations and bone biopsies were performed. The results were compared with those of a previous clinical study that used the original protocol. (3) Results: Panoramic X-ray and computed tomography showed bone regeneration at the transplantation site in all cases. The average bone area in the biopsy samples at 4 months was 44.0%, which was comparable to that in a previous clinical study at 6 months (41.9%) but with much less deviation. No side effects related to cell transplantation were observed. In regenerated bone, 100% of the implants were integrated. (4) Conclusions: Compared to the original protocol, the non-inferiority of this protocol was proven. The introduction of an optimized cell-processing protocol resulted in a comparable quality of regenerated bone, with less fluctuation. Optimized cell-processing protocols may contribute to stable bone regeneration.

Intrathecal Cell Therapy with Autologous Bone Marrow Stromal Cells as a New Tool for Neurologic Sequels after Spinal Cord Surgery: A Report of Two Cases

Background aims: The possibility of permanent neurological sequels after surgery of benign lesions affecting the spinal cord is well known. Frequently, they are irreversible, with no effective treatment other than rehabilitation. However, in recent years, intrathecal cell therapy with autologous bone marrow stromal cells (MSCs) in patients with incomplete paraplegia has shown benefits for diverse sequels of spinal cord injury (SCI). Methods: We present two patients with chronic spinal cord sequels after a surgery, who underwent cell therapy treatment with NC1 medicament (repeated intrathecal administrations of MSCs). Results: In both cases, cell therapy achieved a clear improvement in neurological sequels, such as recovery of gait disturbances, bowel dysfunction, or neuropathic pain. Conclusion: Intrathecal cell therapy with autologous MSCs offers a new approach for neurological sequels after spinal cord surgery.

Clinical Outcome and 8-Year Follow-Up of Alveolar Bone Tissue Engineering for Severely Atrophic Alveolar Bone Using Autologous Bone Marrow Stromal Cells with Platelet-Rich Plasma and β-Tricalcium Phosphate Granules.

Background: Although bone tissue engineering for dentistry has been studied for many years, the clinical outcome for severe cases has not been established. Furthermore, there are limited numbers of studies that include long-term follow-up. In this study, the safety and efficacy of bone tissue engineering for patients with a severely atrophic alveolar bone were examined using autogenous bone marrow stromal cells (BMSCs), and the long-term stability was also evaluated. Methods: BMSCs from iliac bone marrow aspirate were cultured and expanded. Then, induced osteogenic cells were transplanted with autogenous platelet-rich plasma (PRP) and β-tricalcium phosphate granules (β-TCP) for maxillary sinus floor and alveolar ridge augmentation. Eight patients (two males and six females) with an average age of 54.2 years underwent cell transplantation. Safety was assessed by monitoring adverse events. Radiographic evaluation and bone biopsies were performed to evaluate the regenerated bone. Results: The major population of transplanted BMSCs belonged to the fraction of CD34−, CD45dim, and CD73+ cells, which was only 0.065% of the total bone marrow cells. Significant deviations were observed in cell growth and alkaline phosphatase activities among individuals. However, bone regeneration was observed in all patients and the average bone area in the biopsy samples was 41.9% 6 months following transplantation, although there were also significant deviations among each case. No adverse events related to the transplants were observed. In the regenerated bone, 27 out of 29 dental implants were integrated. Dental implants and regenerated bone were stable for an average follow-up period of 7 years and 10 months. Conclusions: Although individual variations were observed, the results showed that bone tissue engineering using BMSCs with PRP and β-TCP was feasible for patients with severe atrophic maxilla throughout a long-term follow-up period and was considered safe. However, further studies with a larger number of cases and controls to confirm the efficacy of BMSCs and the development of a protocol to establish a reproducible quality of stem cell-based graft material will be required.

Selective Retention of Bone Marrow Stromal Cells with Gelatin Sponge for Repair of Intervertebral Disc Defects after Microendoscopic Discectomy: A Prospective Controlled Study and 2-Year Follow-Up.

Objective Discectomy remains the classic procedure for treating lumbar intervertebral disc (IVD) herniation, but the occurrence of defects after discectomy is thought to be an important cause generating recurrent and accelerated IVD degeneration. Previous studies attempted suture of the annulus fissure, but the validity of this technique on restraining the degenerative process is controversial. On the other hand, cell therapies have been shown in multiple clinical and basic studies. Our purpose was to investigate the effectiveness of selective retention of autologous Bone Marrow Stromal Cells (BMSCs) with gelatin sponge in combination with annulus fibrosus suture (AFS) for the repair of IVD defects following mobile microendoscopic discectomy (MMED). Methods This prospective, two-armed, and controlled clinical study was conducted from December 2016 to December 2018. Written informed consent was obtained from each patient. Forty-five patients with typical symptoms, positive signs of radiculopathy, and obvious lumbar disc herniation observed by MRI were enrolled. Patients were divided into 3 groups with different treating methods: MMED (n = 15), MMED+AFS (n = 15), and MMED+AFS+BMSCs (n = 15). A postoperative 2-year follow-up was performed to evaluate the patient-reported outcomes of VAS, ODI, and SF-36. The improvement rate of VAS and ODI was calculated as [(latest‐preoperative)/preoperative] to evaluate the therapeutic effect of the three groups. Assessment parameters included Pfirrmann grade, intervertebral disc height (IDH), and disc protrusion size (DPS), as measured by MRI to evaluate the morphological changes. Results All patients enrolled had a postoperative follow-up at 3, 6, 12, and 24 months. VAS and ODI scores were significantly improved compared to the preoperative status in all three groups with a mean DPS reduction rate over 50%. At the final follow-up, the improvement rate of the VAS score in the MMED+AFS+BMSCs group was significantly higher than the MMED+AFS and MMED groups (80.1% ± 7.6% vs. 71.3% ± 7.0% vs. 70.1% ± 7.8%), while ODI improvement showed a significant change (65.6% ± 8.8% vs. 59.9% ± 5.5% vs. 57.8% ± 8.1%). All participants showed significant improvement in SF-36 PCS and MCS; the differences between each group were not significant. The mean IDH loss rate of the MMED+AFS+BMSCs group was also significantly lower than other groups (−17.2% ± 1.3% vs. −27.6% ± 0.7% vs. −29.3% ± 2.2%). The Pfirrmann grade was aggravated in the MMED and MMED+AFS groups while maintained at the preoperative grade in the MMED+AFS+BMSCs group. No adverse events of cell transplantation or recurrence were found in all patients during the postoperative follow-up period. Conclusions It is feasible and effective to repair lumbar IVD defects using SCR-enriched BMSCs with gelatin sponges, which warrants further study and development as a cell-based therapy for IVD repair.

Stromal Cells Serve Drug Resistance for Multiple Myeloma via Mitochondrial Transfer: A Study on Primary Myeloma and Stromal Cells.

Simple SummaryMitochondrial transfer plays a crucial role in the acquisition of drug resistance in multiple myeloma, but its exact mechanism is not yet clear; moreover, overcoming the drug resistance that it causes is also a major challenge. Our research on primary myeloma cell cultures reveals that mitochondrial transfer is bi-directional between bone marrow stromal cells and myeloma cells, occurring via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, whereupon survival and adenosine triphosphate levels increase, while mitochondrial superoxide levels decrease in myeloma cells. These changes and the elevation of superoxide levels in stromal cells are proportional to the amount of incorporated mitochondria derived from the other cell type and to the concentration of the used drug. Although the inhibition of mitochondrial transfer is limited between stromal and myeloma cells, the supportive effect of stromal cells can be effectively averted by influencing the tumor metabolism with an inhibitor of oxidative phosphorylation in addition to chemotherapeutics.Recently, it has become evident that mitochondrial transfer (MT) plays a crucial role in the acquisition of cancer drug resistance in many hematologic malignancies; however, for multiple myeloma, there is a need to generate novel data to better understand this mechanism. Here, we show that primary myeloma cells (MMs) respond to an increasing concentration of chemotherapeutic drugs with an increase in the acquisition of mitochondria from autologous bone marrow stromal cells (BM-MSCs), whereupon survival and adenosine triphosphate levels of MMs increase, while the mitochondrial superoxide levels decrease in MMs. These changes are proportional to the amount of incorporated BM-MSC-derived mitochondria and to the concentration of the used drug, but seem independent from the type and mechanism of action of chemotherapeutics. In parallel, BM-MSCs also incorporate an increasing amount of MM cell-derived mitochondria accompanied by an elevation of superoxide levels. Using the therapeutic antibodies Daratumumab, Isatuximab, or Elotuzumab, no similar effect was observed regarding the MT. Our research shows that MT occurs via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, but its inhibition is limited. However, the supportive effect of stromal cells can be effectively avoided by influencing the metabolism of myeloma cells with the concomitant use of chemotherapeutic agents and an inhibitor of oxidative phosphorylation.

CD8+ Anti-BCMA mRNA CAR T-Cells Effectively Kill Human Multiple Myeloma Cells In Vitro and In Vivo

Potent anti-multiple myeloma (MM) responses have been observed in early-stage clinical trials with virally-transduced chimeric antigen receptor (CAR) T-cells redirected to B-cell maturation antigen (BCMA), a membrane protein with extremely high sensitivity and specificity for MM and plasma cells. Despite significant potency, CAR T-cells engineered by virally-mediated gene transfer present a poor risk profile. Permanent modification enhances CAR persistence but also increases the risk of severe cytokine release syndrome (CRS), neurotoxicity, and prolonged plasma cell aplasia due to uncontrolled in vivo expansion and long-term persistence of the permanently-modified CAR T-cells. In an effort to address these shortcomings while preserving efficacy, we have developed CD8+ anti-BCMA CAR T-cells modified transiently by mRNA transfection (Descartes-08). Descartes-08 manufactured at clinical scale showed high (>90%) purity, post-cryopreservation viability, and transfection efficiency. Descartes-08 demonstrated robust dose- and time- dependent killing of BCMA+ MM cell lines, including those with acquired resistance to immunomodulatory drugs (IMiDs), as well as primary MM cells co-cultured with autologous bone marrow stromal cells. Compared with transfected pan- (CD3+) anti-BCMA CAR T-cells, Descartes-08 (CD8+) demonstrated enhanced transfection and killing but reduced secretion of IFN-g, an inflammatory cytokine highly correlated with CRS. Descartes-08 also demonstrated robust dose-dependent efficacy in a disseminated human MM model (i.v. MM1SLuc cells in NSG mice), and could be safely administered repetitively to deepen responses. These results advocate for rapid clinical translation of Descartes-08, which may improve the benefit: risk profile of CAR T-cells in MM and enable their use in patients with earlier-stage disease. DisclosuresKurtoglu:2Cartesian Therapeutics, Inc: Employment, Equity Ownership. Holderness:Cartesian Therapeutics, Inc: Employment, Equity Ownership. Kalayoglu:Cartesian Therapeutics, Inc: Employment, Equity Ownership. Richardson:Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees. Anderson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder.

Articular cartilage regeneration in rats using human bone marrow stromal cells

Purpose: Injury to articular cartilage found in knee joints is often disabling and affects both younger and older individuals. Injured cartilage rarely heals, leading to a progressive loss of normal function of the joint in many cases. Current treatments are far from satisfactory. One treatment is surgery (such as drilling through the cartilage to the bone, abrasion to remove diseased cartilage and microfracture). These procedures can result in the formation of a mixture of fibrous tissue and cartilage within the injury, which does not lead to a total recovery of joint function. Autologous human bone marrow stromal cell (hBMSC) transplantation maybe a potential avenue to regenerate articular cartilage. We previously determined that naive (untreated) hBMSC were able to form stable, non-hypertrophic cartilage when transplanted subcutaneously in conjunction with fibrin microbeads covalently coated with hyaluronic acid (FMBs). In our current study, we are investigating the ability of hBMSC/FMB constructs to generate native-like cartilage in an articular defect in immunocompromised rodents. Methods: hBMSCs were obtained from surgical bone waste and grown in culture for 2-3 passages. Approval for this study was given by the institutional Animal Care and Use Committee (ACUC). To create the articular cartilage defects in immunocompromised SRG rats (Hera/Charles River), the patella was laterally displaced and the distal femur was immobilized using a clamp. The defect was made in the trochlear groove of the distal femur using a 1.8mm diameter microdrill. The defect was either left unrepaired (sham), or subsequently filled with either FMBs alone (FMBs Alone), hBMSCs attached to FMBs (FMBs+cells), or with a pellet created by incubating BMSCs with FMBs for 10 days in chondrogenic medium. At the 4-week and 8-week timepoints following surgery, the rats were euthanized and femurs were harvested and fixed in 4% paraformaldehyde for 24 hours and then decalcified using a 10% EDTA solution for 30 days. After decalcification, the femurs were paraffin embedded and sectioned at a thickness of 6 μm. Histological staining was performed using hematoxylin and eosin, toluidine blue, and safranin O methods to assess cartilage formation. Immunohistochemistry for aggrecan within the cartilage was also performed to assess cartilage proteoglycan formation. Image analysis using Image J was performed on sections stained with safranin O. Cartilage thickness was approximated based on the thickness of the intact cartilage surrounding the defect, and a region of interest (ROI) was drawn surrounding the defect with the same depth as the intact cartilage. The image color channels were separated into red, green, and blue, and the blue channel was used to threshold for the stained proteoglycan. The “analyze particles” module of Image J was used to calculate the percentage of the ROI that stained positive for proteoglycan. Second-harmonic generation microscopy was also used to evaluate collagen fibril structure and organization. Results: Toluidine blue staining revealed cartilage formation in defects at the 4-week and 8-week timepoints in both the FMBs+cells and pellet groups. No cartilage was detected in the FMBs alone or sham groups (Figure 1). Percentages of defect area positive for cartilage proteoglycan in the FMBs+cells, pellet, FMBs alone, and sham group defect areas were 8.95%, 3.03%, 0.008%, and 0.094% respectively. A comparable ROI in healthy rat cartilage showed proteoglycan positivity of 58.43% of the area (Figure 2). Second harmonic generation imaging of the defect area showed that from sham to FMB alone to pellet to FMBs+cells groups, the organization of the collagen fibrils begin to more closely approximate healthy rat cartilage (Figure 3). Long-term time points are currently being analyzed. Conclusions: At the early timepoints presented in this abstract, there is evidence of cartilage formation in the drilled articular cartilage defects in both the FMBs+cells and pellet groups. This initial data shows the potential for our method to regenerate articular cartilage in vivo. Analysis of our long-term time points will reveal the extent and quality of healing induced by hBMSCs transplanted in conjunction with FMBs.

Экспериментальное обоснование клинического применения стимуляторов остеогенеза в травматологии и ортопедии (обзор литературы)

Analysis of scientifi c publications devoted to effi cacy assessment and experimental justifi cation of cell technology and growth factors application for osteogenesis stimulation in clinical practice of traumatologists-orthopaedists.The search was performed for papers published in PubMed and E-library digital databases within the period of 2010-2020. The search filter was set to explore randomised clinical trials. Th e key words were: “platelet growth factor OR vascular endothelial growth factor OR bone morphogenetic proteins OR autologous bone marrow stromal cells AND bone defect AND human”. Works dated 2010-2020 were selected for analysis of experimental studies using the following key word combinations: “mesenchymal stromal cells AND osteogenesis OR bone regeneration”, “growth factors AND mesenchymal stromal cells AND osteogenesis OR bone regeneration”. In addition, essential literatures describing properties of multipotent mesenchymal stromal cells (MSCs) were cited as well as issues in their osteogenic diff erentiation. Despite the great number of experimental works related to cell technology application for bone growth stimulation, a large variety of unresolved issues associated with optimal means to create such cells, their cultivation and combined application with morphogenetic proteins and growth factors remains. Clinical application of rhBMP-2, PDGF has demonstrated effi cacy in complex treatment of patients with bone defects. Th erewith, growth factors may be effi caciously used in diff erent combinations with MSCs. In a number of cases, application of implants enriched with growth factors may become an alternative for autogenic grafts. To the present day, there is crucial defi cit in the number of works presenting results of randomised controlled clinical trials devoted to stimulation of bone tissue repair. The controversial results of the publications analysed may be based on a low number of patients enrolled. With consideration for active development of reparative medicine as a whole, further investigation of possibilities in bone growth stimulation with local application of cell-engineering constructions is considered to be a promising trend for traumatology and orthopaedics.

Autologous Endothelial Progenitor Cells Transplantation for Acute Ischemic Stroke: A 4-Year Follow-Up Study.

Transplantation of endothelial progenitor cells (EPCs) is a proven safe and effective method for treatment of cerebral ischemia in animal experiments. However, safety and efficacy need to be determined in clinical trials. We performed a two‐center, randomized, placebo‐controlled phase I/IIa trial with blinded outcome assessment on 18 patients with acute cerebral infarct within the middle cerebral artery territory, and followed for up to 4 years. Autologous ex vivo expanded EPCs were injected intravenously in the EPC group, and patients who received saline or autologous bone marrow stromal cells served as control groups. Mortality of any cause, adverse events, and new‐onset comorbidities were monitored. Changes in neurological deficits were assessed at different time points. We found no toxicity events or infusional or allergic reactions in any treated group. Three patients in the placebo group died during the 4‐year follow‐up. We found that the EPC group had fewer serious adverse events compared with the placebo‐controlled group, although there were no statistical differences in mortality among the three groups. Furthermore, there was no significant difference in neurological or functional improvement observed among the three groups, except for the Scandinavia Stroke Scale score at 3 months between the EPC group and placebo‐controlled group. Autologous transplantation of EPCs appears to improve long‐term safety in acute cerebral infarct patients, supporting the feasibility of this novel method for treatment of ischemic stroke (ClinicalTrials.gov: NCT01468064). Stem Cells Translational Medicine 2019;8:14–21

Revitalized and synovialized allograft for intrasynovial flexor tendon reconstruction in an in vivo canine model.

This study was to test our hypothesis that flexor tendon reconstruction with an allograft revitalized with bone marrow stromal cells (BMSCs) and synovialized with carbodiimide derivatized autologous synovial fluid (cd-SYN) would result in better digit functional restoration than the conventional allograft tendon. A total of 32 flexor digital profundus tendons from the second and fifth digit of 16 dogs were created a repair failure model first. Then, failed-repaired tendons were reconstructed with either a revitalized-synovialized allograft tendon or a clinical standard autograft tendon (control group). The allograft tendon was seeded with autologous BMSCs in multiple slits and the graft surface was coated with cd-SYN. A 6 weeks after tendon reconstruction, the digits were harvested and evaluated for digit function, adhesion status, tendon gliding resistance, attachment strength, cell viability, and histologic factors. The allograft group had significantly improved digit function compared with the control group through decreased work of flexion, increased digit range of motion under 2-Newton force, and less adhesion score (p < .05). However, the distal attachment-site strength and stiffness in the allograft tendon were significantly weaker than the autografts (p < .05). No significant difference was found for gliding resistance. Histologically, allograft tendons coated with allograft had smoother surfaces and showed tendon-to-bone and tendon-to-tendon incorporation. Viable BMSCs were found in the tendon slits 6 weeks after the graft. In conclusion, cellular lubricant-based modification of allograft tendons improved digit function and reduced the adhesions compared with autograft for flexor tendon reconstruction. However, improvement of graft-to-host tendon healing is still challenging. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Intrathecal administration of autologous bone marrow stromal cells improves neuropathic pain in patients with spinal cord injury

Neuropathic pain (NP) is highly disabling, responds poorly to pharmacological treatment, and represents a significant cause of decreased quality of life in patients suffering from spinal cord injury (SCI). In recent years, cell therapy with autologous mesenchymal stromal cells (MSCs) has been considered as a potential therapeutic weapon in this entity. Ten patients suffering chronic SCI received 100 million MSCs into subarachnoid space by lumbar puncture (month 1 of the study) and this procedure was repeated at months 4 and 7 until reaching a total doses of 300 million MSCs. Intensity of NP was measured by standard numerical rating scale (VAS) from 0 to 10, recording scores previous to the first MSCs administration and monthly, until month 10 of follow-up. Months 1, 4, 7 and 10 of the study were selected as time points in order to a statistical analysis by the nonparametric Wilcoxon rank test. Our results showed significant and progressive improvement in NP intensity after the first administration of MSCs (p: 0.003). This study supports the benefit of intrathecal administration of autologous MSCs for the treatment of NP in patients with SCI.

Repair of articular cartilage defects with acellular cartilage sheets in a swine model

Acellular cartilage sheets (ACSs) have been demonstrated as a good biomaterial for cartilage regeneration as a result of their natural cartilage matrix components, cartilage-specific structures, and good biocompatibility. However, it remains unknown whether allogeneic ACSs could promote cartilage regeneration and repair cartilage defects in a large animal model. The current study explored the feasibility of repairing articular cartilage defects using ACS scaffold with or without autologous bone marrow stromal cells (BMSCs) in a swine model. According to the current results, ACSs retained natural cartilage structure, primary cartilage matrices, and cartilage-specific growth factors. After cell seeding, ACSs presented good biocompatibility with BMSCs, which produced abundant extracellular matrix (ECM) proteins to cover the lacuna structures. In vivo results indicated that ACSs alone could induce endogenous host cells to regenerate cartilage and achieve generally satisfactory repair of cartilage defects at 6 months post-operation, including good interface integration and cartilage-specific ECM deposition. After combination with autologous BMSCs, BMSC-ACS constructs achieved more satisfactory repair of cartilage defects even without in vitro pre-induction of chondrogenesis. More importantly, all defects in both BMSC-ACS and ACS-only groups showed enhanced cartilage regeneration compared with BMSC–polyglycolic acid and blank groups, which mainly exhibited fibrogenesis in defect areas. Collectively, the current results indicate that ACSs can efficiently repair articular cartilage defects by promoting chondrogenic differentiation of BMSCs or inducing endogenous chondrogenesis in situ, thus serving as a good cartilage regeneration scaffold for recovery of articular function.

Three dimensional printed polylactic acid-hydroxyapatite composite scaffolds for prefabricating vascularized tissue engineered bone: An in vivo bioreactor model

The repair of large bone defects with complex geometries remains a major clinical challenge. Here, we explored the feasibility of fabricating polylactic acid-hydroxyapatite (PLA-HA) composite scaffolds. These scaffolds were constructed from vascularized tissue engineered bone using an in vivo bioreactor (IVB) strategy with three-dimensional printing technology. Specifically, a rabbit model was established to prefabricate vascularized tissue engineered bone in two groups. An experimental group (EG) was designed using a tibial periosteum capsule filled with 3D printed (3DP) PLA-HA composite scaffolds seeded with bone marrow stromal cells (BMSCs) and crossed with a vascular bundle. 3DP PLA-HA scaffolds were also combined with autologous BMSCs and transplanted to tibial periosteum without blood vessel as a control group (CG). After four and eight weeks, neovascularisation and bone tissues were analysed by studying related genes, micro-computed tomography (Micro-CT) and histological examinations between groups. The results showed that our method capably generated vascularized tissue engineered bone in vivo. Furthermore, we observed significant differences in neovascular and new viable bone formation in the two groups. In this study, we demonstrated the feasibility of generating large vascularized bone tissues in vivo with 3DP PLA-HA composite scaffolds.

Research on advanced intervention using novel bone marrOW stem cell (RAINBOW): a study protocol for a phase I, open-label, uncontrolled, dose-response trial of autologous bone marrow stromal cell transplantation in patients with acute ischemic stroke

BackgroundStroke is a leading cause of death and disability, and despite intensive research, few treatment options exist. However, a recent breakthrough in cell therapy is expected to reverse the neurological sequelae of stroke. Although some pioneer studies on the use of cell therapy for treating stroke have been reported, certain problems remain unsolved. Recent studies have demonstrated that bone marrow stromal cells (BMSCs) have therapeutic potential against stroke. We investigated the use of autologous BMSC transplantation as a next-generation cell therapy for treating stroke. In this article, we introduce the protocol of a new clinical trial, the Research on Advanced Intervention using Novel Bone marrOW stem cell (RAINBOW).Methods/designRAINBOW is a phase 1, open-label, uncontrolled, dose-response study, with the primary aim to determine the safety of the autologous BMSC product HUNS001–01 when administered to patients with acute ischemic stroke. Estimated enrollment is 6–10 patients suffering from moderate to severe neurological deficits. Approximately 50 mL of the bone marrow is extracted from the iliac bone of each patient 15 days or later from the onset. BMSCs are cultured with allogeneic human platelet lysate (PL) as a substitute for fetal calf serum and are labeled with superparamagnetic iron oxide for cell tracking using magnetic resonance imaging (MRI). HUNS001–01 is stereotactically administered around the area of infarction in the subacute phase. Each patient will be administered a dose of 20 or 50 million cells. Neurological scoring, MRI for cell tracking, 18F–fuorodeoxyglucose positron emission tomography, and 123I–Iomazenil single­photon emission computed tomography will be performed for 1 year after the administration.DiscussionThis is a first-in-human trial for HUNS001–01 to the patients with acute ischemic stroke. We expect that intraparenchymal injection can be a more favorable method for cell delivery to the lesion and improvement of the motor function than intravenous infusion. Moreover, it is expected that the bio-imaging techniques can clarify the therapeutic mechanisms.Trial registrationThe trial was registered at The University Hospital Medical Information Network on February 22, 2017 (UNIN ID: UMIN000026130). The findings of this trial will be disseminated to patients and through peer-reviewed publications and international presentations.