Circ_0008542 in osteoblast exosomes promotes osteoclast-induced bone resorption through m6A methylation.

With an increasing aging society, China is the world's fastest growing markets for oral implants. Compared with traditional oral implants, immediate implants cause marginal bone resorption and increase the failure rate of osseointegration, but the mechanism is still unknown. Therefore, it is important to further study mechanisms of tension stimulus on osteoblasts and osteoclasts at the early stage of osseointegration to promote rapid osseointegration around oral implants. The results showed that exosomes containing circ_0008542 from MC3T3-E1 cells with prolonged tensile stimulation promoted osteoclast differentiation and bone resorption. Circ_0008542 upregulated Tnfrsf11a (RANK) gene expression by acting as a miR-185-5p sponge. Meanwhile, the circ_0008542 1916-1992 bp segment exhibited increased m6A methylation levels. Inhibiting the RNA methyltransferase METTL3 or overexpressing the RNA demethylase ALKBH5 reversed osteoclast differentiation and bone resorption induced by circ_0008542. Injection of circ_0008542 + ALKBH5 into the tail vein of mice reversed the same effects in vivo. Site-directed mutagenesis study demonstrated that 1956 bp on circ_0008542 is the m6A functional site with the abovementioned biological functions. In conclusion, the RNA methylase METTL3 acts on the m6A functional site of 1956 bp in circ_0008542, promoting competitive binding of miRNA-185-5p by circ_0008542, and leading to an increase in the target gene RANK and the initiation of osteoclast bone absorption. In contrast, the RNA demethylase ALKBH5 inhibits the binding of circ_0008542 with miRNA-185-5p to correct the bone resorption process. The potential value of this study provides methods to enhance the resistance of immediate implants through use of exosomes releasing ALKBH5.

Comparative analysis of plasma-derived albumin scaffold, alveolar osteoblasts and synthetic membrane in critical mandibular bone defects: An experimental study on rats.

Repair of bone deficiencies in the craniofacial skeleton remains a challenging clinical problem. The aim of this study was to evaluate and compare the effects of a plasma-derived albumin scaffold, alveolar osteoblasts and synthetic membrane implanted into experimental mandibular defects. Bilateral mandibular defects were created in twelve immunodeficient rats. The bone defect was filled with serum scaffold alone in left sides and scaffold combined with human alveolar osteoblast in right side defects. Implanted areas were closed directly in Group 1 (n = 6) and covered by a resorbable polyglycolic-polylactic acid membrane in Group 2 (n = 6). Bone regeneration was determined at 12 weeks as measured by and exhaustive multiplanar computed tomography analysis and histological examination. No significant differences in bone density were observed between defects transplanted with scaffold alone or scaffold seeded with osteoblasts. The use of membrane did not result in a determining factor in the grade of bone regeneration between Groups 1 and 2. Based on these results, it could be concluded that the albumin scaffold alone has osteoinductive capacity but presence of seeded ostogenic cells accelerates defect repair without being significantly influenced by covering the defect with a resorbable membrane.

The Progress of Stem Cell Technology for Skeletal Regeneration.

Skeletal disorders, such as osteoarthritis and bone fractures, are among the major conditions that can compromise the quality of daily life of elderly individuals. To treat them, regenerative therapies using skeletal cells have been an attractive choice for patients with unmet clinical needs. Currently, there are two major strategies to prepare the cell sources. The first is to use induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), which can recapitulate the skeletal developmental process and differentiate into various skeletal cells. Skeletal tissues are derived from three distinct origins: the neural crest, paraxial mesoderm, and lateral plate mesoderm. Thus, various protocols have been proposed to recapitulate the sequential process of skeletal development. The second strategy is to extract stem cells from skeletal tissues. In addition to mesenchymal stem/stromal cells (MSCs), multiple cell types have been identified as alternative cell sources. These cells have distinct multipotent properties allowing them to differentiate into skeletal cells and various potential applications for skeletal regeneration. In this review, we summarize state-of-the-art research in stem cell differentiation based on the understanding of embryogenic skeletal development and stem cells existing in skeletal tissues. We then discuss the potential applications of these cell types for regenerative medicine.

Osteoblast-Based Therapy-A New Approach for Bone Repair in Osteoporosis: Pre-Clinical Setting.

BACKGROUND: Osteoporosis is a metabolic bone disease characterized by low bone density resulting in increased fracture susceptibility. This research was constructed to uncover the potential therapeutic application of osteoblasts transplantation, generated upon culturing male rat bone marrow-derived mesenchymal stem cells (BM-MSCs) in osteogenic medium (OM), OM containing gold (Au-NPs) or gold/hydroxyapatite (Au/HA-NPs) nanoparticles, in ovariectomized rats to counteract osteoporosis. METHODS: Forty rats were randomized into: (1) negative control, (2) osteoporotic rats, whereas groups (3), (4) and (5) constituted osteoporotic rats treated with osteoblasts yielded from culturing BM-MSCs in OM, OM plus Au-NPs or Au/HA-NPs, respectively. After 3 months, osterix (OSX), bone alkaline phosphatase (BALP), sclerostin (SOST) and bone sialoprotein (BSP) serum levels were assessed. In addition, gene expression levels of cathepsin K, receptor activator of nuclear factor-κb ligand (RANKL), osteoprotegerin (OPG) and RANKL/OPG ratio were evaluated using real-time PCR. Moreover, histological investigation of femur bone tissues in different groups was performed. The homing of implanted osteoblasts to the osteoporotic femur bone of rats was documented by Sex determining region Y gene detection in bone tissue. RESULTS: Our results indicated that osteoblasts infusion significantly blunted serum BALP, BSP and SOST levels, while significantly elevated OSX level. Also, they brought about significant down-regulation in gene expression levels of cathepsin K, RANKL and RANKL/OPG ratio versus untreated osteoporotic rats. Additionally, osteoblasts nidation could restore bone histoarchitecture. CONCLUSION: These findings offer scientific evidence that transplanting osteoblasts in osteoporotic rats regains the homeostasis of the bone remodeling cycle, thus providing a promising treatment strategy for primary osteoporosis.

Uptake of osteoblast-derived extracellular vesicles promotes the differentiation of osteoclasts in the zebrafish scale.

Differentiation of osteoclasts (OCs) from hematopoietic cells requires cellular interaction with osteoblasts (OBs). Due to the difficulty of live-imaging in the bone, however, the cellular and molecular mechanisms underlying intercellular communication involved in OC differentiation are still elusive. Here, we develop a fracture healing model using the scale of trap:GFP; osterix:mCherry transgenic zebrafish to visualize the interaction between OCs and OBs. Transplantation assays followed by flow cytometric analysis reveal that most trap:GFPhigh OCs in the fractured scale are detected in the osterix:mCherry+ fraction because of uptake of OB-derived extracellular vesicles (EVs). In vivo live-imaging shows that immature OCs actively interact with osterix:mCherry+ OBs and engulf EVs prior to convergence at the fracture site. In vitro cell culture assays show that OB-derived EVs promote OC differentiation via Rankl signaling. Collectively, these data suggest that EV-mediated intercellular communication with OBs plays an important role in the differentiation of OCs in bone tissue.

Three-dimensional periodontal tissue regeneration using a bone-ligament complex cell sheet.

Periodontal tissue is a distinctive tissue structure composed three-dimensionally of cementum, periodontal ligament (PDL) and alveolar bone. Severe periodontal diseases cause fundamental problems for oral function and general health, and conventional dental treatments are insufficient for healing to healthy periodontal tissue. Cell sheet technology has been used in many tissue regenerations, including periodontal tissue, to transplant appropriate stem/progenitor cells for tissue regeneration of a target site as a uniform tissue. However, it is still difficult to construct a three-dimensional structure of complex tissue composed of multiple types of cells, and the transplantation of a single cell sheet cannot sufficiently regenerate a large-scale tissue injury. Here, we fabricated a three-dimensional complex cell sheet composed of a bone-ligament structure by layering PDL cells and osteoblast-like cells on a temperature responsive culture dish. Following ectopic and orthotopic transplantation, only the complex cell sheet group was demonstrated to anatomically regenerate the bone-ligament structure along with the functional connection of PDL-like fibers to the tooth root and alveolar bone. This study represents successful three-dimensional tissue regeneration of a large-scale tissue injury using a bioengineered tissue designed to simulate the anatomical structure.

Did Osteoblastic Cell Therapy Improve the Prognosis of Pre-fracture Osteonecrosis of the Femoral Head? A Randomized, Controlled Trial.

BACKGROUND: In patients with nontraumatic osteonecrosis of the femoral head (ONFH), implantation of bone marrow aspirate concentrate (BMAC) could delay the progression of osteonecrosis and improve symptoms in pre-fracture ONFH. However, the BMAC content, especially in osteoblastic stem cells, could have an important individual variability. An autologous osteoblastic cell product could improve the effect of such cell-based therapy. QUESTIONS/PURPOSES: (1) Does autologous osteoblastic cell therapy decrease the likelihood of progression to subchondral fracture with or without early collapse corresponding to Association Research Circulation Osseous (ARCO) classification Stage III or higher, and provide a clinically important pain improvement compared with BMAC treatment alone? (2) Were patients treated with osteoblastic cell therapy less likely to undergo subsequent THA? (3) What proportion of patients in the treatment and control groups experienced adverse events after surgery? METHODS: Between 2004 and 2011, we treated 279 patients for Stage I to II hip osteonecrosis (ON) with surgery. During that time, our general indications for surgery in this setting included non-fracture ON lesions. To be eligible for this randomized, single-blind trial, patients needed to have an ONFH Stage I or II; we excluded those with traumatic ONFH, hemoglobinopathies and positive serology for hepatitis B, C or HIV. Of those treated surgically for this diagnosis during the study period, 24% (67) agreed to participate in this randomized trial. Hips with pre-fracture ONFH were randomly treated with a core decompression procedure associated with either implantation of a BMAC (BMAC group; n = 26) or osteoblastic cell (osteoblastic cell group; n = 30). The groups were not different in terms of clinical and imaging characteristics. The primary study outcome was treatment response, defined as the absence of progression to subchondral fracture stage (ARCO stage III or higher) plus a clinically important pain improvement defined as 1 cm on a 10-cm VAS. The secondary endpoint of interest was the frequency in each group of subsequent THA and the frequency of adverse events. The follow-up duration was 36 months. We used an as-treated analysis (rather than intention-to-treat) for our efficacy endpoint, and an intention-to-treat analysis for adverse events. Overall, 26 of 26 patients in the BMAC group and 27 of 30 in the osteoblastic cell group completed the trial. RESULTS: At 36 months, no clinically important differences were found in any study endpoint. There was no difference in the proportion of patients who had progressed to fracture (ARCO stage III or higher; 46% of the BMAC hips [12 of 26] versus 22% in the hips with osteoblastic cells [six of 27], hazard ratio, 0.47 [95% CI 0.17 to 1.31]; p = 0.15). There was no clinically important difference in VAS pain scores. No differences were found for either the WOMAC or the Lequesne indexes. With the numbers available, there was no difference in the proportion of patients in the groups who underwent THA at 36 months 15% (four of 27) with osteoblastic cells versus 35% (nine of 26) with BMAC; p = 0.09 With the numbers available, we found no differences between the treatment and control groups in terms of the frequencies of major adverse events. CONCLUSIONS: We found no benefit to osteoblastic cells over BMAC in patients with pre-collapse ONFH; side effects were uncommon and generally mild in both groups. This study could be used as pilot data to help determine sample sizes for larger (presumably multicenter) randomized controlled trials. However, this novel treatment cannot be recommended in routine practice until future, larger studies demonstrate efficacy. LEVEL OF EVIDENCE: Level II, therapeutic study.

Cross-linked gelatin microsphere-based scaffolds as a delivery vehicle of MC3T3-E1 cells: in vitro and in vivo evaluation.

Scaffolding plays a crucial role in bone tissue engineering by not only providing interfaces for cell adhesion, proliferation, and differentiation but also guiding neotissue formation. For this purpose, microspheres (MSs) are being increasingly used alone or in combination with other scaffolds. However, few researchers have used MSs to prepare 3D scaffolds by culture with delivered cells. In this study, we have developed covalent cross-linked gelatin MSs (ccG-MSs) (average diameter = 100-300 µm) to load mouse osteoblast MC3T3-E1 cells, which exhibit attachment and spreading on surfaces of ccG-MSs after co-culture. Significantly, the ccG-MSs can be integrated into a macroscopic construct with MC3T3-E1 cells after 5 days of cultivation. The MC3T3-E1 cells within ccG-MSs constructs show a higher viability and proliferation activity than those in the micro-cavitary gelatin gel (MCG) constructs. Calcium deposition, alkaline phosphatase activity as well as osteocalcin secretion within both ccG-MSs and MCG constructs have been evaluated in vitro and in vivo, respectively. Compared to MCG scaffolds, ccG-MS-based scaffolds can provide better cellular microenvironments for cell proliferation and osteogenic differentiation. Our findings will lay the foundation for understanding cellular behaviors in MS-based 3D constructs and help in designing MS-based bone tissue engineering scaffolds.

An osteoconductive PLGA scaffold with bioactive ß-TCP and anti-inflammatory Mg(OH)2 to improve in vivo bone regeneration.

Poly(lactic-co-glycolic acid) (PLGA) has been widely used as a biomaterial for pharmaceutical and medical applications. However, the decomposition products of PLGA are known to acidify the surrounding tissue of the implanted site, causing an inflammatory response. Previously, we developed PLGA/inorganic nanocomposites and optimized the amounts of inorganic compounds, ß-tricalcium phosphate (ß-TCP) and magnesium hydroxide [Mg(OH)2], in terms of osteogenesis of normal human osteoblasts and anti-inflammatory responses of preosteoclastic cells in vitro. In this study, the potential of the optimized PLGA/ß-TCP/Mg(OH)2 nanocomposite (TCP/MH) to promote bone repair through osteoinductive, osteoconductive, and anti-inflammatory abilities was assessed using a bone defect in a rat humeral defect model. PLGA nanocomposites with or without inorganic compounds, PLGA, ß-TCP, MH, and TCP/MH were prepared through one-step bulk modification using a twin-screw extruder. The resulting TCP/MH nanocomposite successfully enhanced the bone regeneration rate for allowing complete bone defect healing with significantly suppressed inflammatory responses. Taken together, the organic and inorganic bioactive nanocomposite developed in this study, TCP/MH, is a promising material in orthopedic implantation.

Enzymatically gellable gelatin improves nano-hydroxyapatite-alginate microcapsule characteristics for modular bone tissue formation.

To maintain gelatin (Gel) as adhesive motifs inside alginate microcapsule as building blocks of modular approach, phenol moiety (Ph) was introduced into gelatin (Gel Ph). Addition of Gel Ph to alginate (Alg-Gel Ph) dramatically altered the physical properties of alginate-based hydrogels as compared to unmodified gelatin (Alg-Gel) addition. Alg-Gel Ph hydrogels revealed a dramatically lower swelling ratios (63%) as compared to Alg-Gel hydrogels (150%). Moreover, Gel Ph decreased 40% degradation rate of alginate-based hydrogels after 72 hr, while increasing compressive modulus 3.5-fold as compared to Alg-Gel hydrogels. Introducing nano-hydroxyapatite (nHA) to Alg-Gel Ph hydrogel (Alg-Gel Ph-nHA) could reduce degradation rate to 41.5% and improve compressive modulus of hydrogels significantly, reaching to 294 ± 2.5 kPa. The microencapsulated osteoblast-like cells proliferated considerably and showed more metabolic activities (two times) in Alg-Gel Ph-nHA microcapsules during a 21-day culture period, resulting in more calcium deposition and alkaline phosphatase (ALP) activities. The subcutaneous microcapsules could also be identified readily without complete absorption and signs of toxicity or any untoward reactions and viable osteoblast-like cells were seen as red colored areas in the central regions of cell-laden microcapsules after 1 month. The study demonstrated Alg-Gel Ph-nHA microcapsule as a promising 3D microenvironment for modular bone tissue formation.

Cytoprotective Preconditioning of Osteoblast-Like Cells with N-Acetyl-L-Cysteine for Bone Regeneration in Cell Therapy.

Oxidative stress hinders tissue regeneration in cell therapy by inducing apoptosis and dysfunction in transplanted cells. N-acetyl-L-cysteine (NAC) reinforces cellular antioxidant capabilities by increasing a major cellular endogenous antioxidant molecule, glutathione, and promotes osteogenic differentiation. This study investigates the effects of pretreatment of osteoblast-like cells with NAC on oxidative stress-induced apoptosis and dysfunction and bone regeneration in local transplants. Rat femur bone marrow-derived osteoblast-like cells preincubated for 3 h with and without 5 mM NAC were cultured in a NAC-free osteogenic differentiation medium with continuous exposure to 50 µM hydrogen peroxide to induce oxidative stress. NAC preincubation prevented disruption of intracellular redox balance and alleviated apoptosis and negative impact on osteogenic differentiation, even under oxidative stress. Autologous osteoblast-like cells with and without NAC pretreatment in a collagen sponge vehicle were implanted in critical-size defects in rat femurs. In the third week, NAC-pretreated cells yielded complete defect closure with significantly matured lamellar bone tissue in contrast with poor bone healing by cells without pretreatment. Cell-tracking analysis demonstrated direct bone deposition by transplanted cells pretreated with NAC. Pretreatment of osteoblast-like cells with NAC enhances bone regeneration in local transplantation by preventing oxidative stress-induced apoptosis and dysfunction at the transplanted site.

Co-culture of the bone and bone marrow: a novel way to obtain mesenchymal stem cells with enhanced osteogenic ability for fracture healing in SD rats.

BACKGROUND: Mesenchymal stem cells (MSCs) have great potential for the repair and regeneration of bone fracture, but their optimal origins remain controversial. METHODS: Bone marrow-MSCs (BM-MSCs) and bone-bone marrow-MSCs (B-BM-MSCs) were isolated from 12 SD rats, and the morphology, MSC-associated markers, and proliferative capacity of these cells were compared using an inverted microscope, flow cytometry, and CCK-8 assays, respectively. After 14 days of osteoblastic induction, osteoblast phenotypes were detected by ALP and calcium nodule staining, and the expression of BMP-2 and TGF-ß1 was observed by western blotting. Then, the rat tibia fracture model was established with 3 groups (n = 6 per group), the control, BM-MSC, and B-BM-MSC groups. Computed tomography (CT) imaging was performed to evaluate fracture healing at weeks 2, 4, and 6. Finally, the fractured bones were removed at weeks 4 and 6, and HE staining was performed to evaluate fracture healing. RESULTS: Although the 2 types of MSCs shared the same cellular morphology and MSC-associated markers, B-BM-MSCs had a higher proliferative rate than BM-MSCs from day 9 to day 12 (p < 0.05), and the expression levels of ALP and calcium were obviously higher in B-BM-MSCs than in BM-MSCs after osteogenic induction (p < 0.01 and p < 0.001, respectively). Western blot results showed that the expression levels of BMP-2 and TGF-ß1 in B-BM-MSCs were higher than in BM-MSCs before and after osteogenic induction (p < 0.01). In the animal experiments, CT imaging and gross observation showed that B-BM-MSCs had a greater capacity than BM-MSCs to promote fracture healing, as the Lane-Sandhu scores of B-BM-MSCs at weeks 4 and 6 after operation (3.00 ± 0.81 and 9.67 ± 0.94, respectively) were higher than those of BM-MSCs (1.33 ± 0.47 and 6.67 ± 1.25, respectively; both p < 0.05). The HE staining results further supported this conclusion. CONCLUSIONS: Taken together, our study results proved that MSCs obtained by co-culturing the bone and bone marrow from SD rats had better proliferative, osteogenic differentiation, and fracture healing capacities than BM-MSCs, perhaps suggesting a novel way to obtain MSCs for bone tissue repair.

Interplay between microRNAs and Wnt, transforming growth factor-ß, and bone morphogenic protein signaling pathways promote osteoblastic differentiation of mesenchymal stem cells.

Osteoblasts are terminally differentiated cells with mesenchymal origins, known to possess pivotal roles in sustaining bone microstructure and homeostasis. These cells are implicated in the pathophysiology of various bone disorders, especially osteoporosis. Over the last few decades, strategies to impede bone resorption, principally by bisphosphonates, have been mainstay of treatment of osteoporosis; however, in recent years more attention has been drawn on bone-forming approaches for managing osteoporosis. MicroRNAs (miRNAs) are a broad category of noncoding short sequence RNA fragments that posttranscriptionally regulate the expression of diverse functional and structural genes in a negative manner. An accumulating body of evidence signifies that miRNAs direct mesenchymal stem cells toward osteoblast differentiation and bone formation through bone morphogenic protein, transforming growth factor-ß, and Wnt signaling pathways. MiRNAs are regarded as excellent future therapeutic candidates because of their small size and ease of delivery into the cells. Considering their novel therapeutic significance, this review discusses the main miRNAs contributing to the anabolic aspects of bone formation and illustrates their interactions with corresponding signaling pathways involved in osteoblastic differentiation.

Silica/polycaprolactone nanofiber scaffold variants for human periosteal cell growth.

Polycaprolactone (PCL) nanofiber scaffolds with attached cadaveric human periosteum or its cells were investigated in this study as a tissue-engineering approach to repair nonunion injuries of bone. Addition of silica nanoparticles (silica or nSiO2 ) to PCL scaffolds was examined for effects on the growth of human periosteal cells in vitro and in vivo. Electrospun PCL nanofiber (nanoPCL) scaffolds were fabricated with different silica contents (0, 0.5, and 1.0 wt %) and utilized as substrates on which periosteal cells were seeded. Human periosteal cell growth analyzed in vitro over 21 days with a PrestoBlue viability assay increased as a function of culture time on each of the three different silica/nanoPCL scaffolds. Cadaveric periosteum attached to nanoPCL scaffolds with or without silica was wrapped around allograft bone and implanted for 10 or 20 weeks in athymic (nude) mice. Histological and immunohistochemical analyses of these experiments in vivo confirmed the presence of viable cells populating the constructs after their retrieval from host mice. Osterix, a marker for osteoblasts, increased in retrieved constructs over time and indicated remodeling of the underlying allograft bone. Summary results suggest that silica/nanoPCL scaffolds may be utilized as substrates for periosteal cell and tissue expansion to augment and support clinical applications for treatment and healing of bone defects, including segmental bone injuries and nonunions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 791-801, 2019.

Enriching Stem/Progenitor Cells from Dental Pulp Cells by Low-density Culturing.

BACKGROUND/AIM: Clonogenicity is a key feature of stem/progenitor cells. The present study aimed to enrich stem/progenitor cells from dental pulp cells by means of culturing the cells at a low clonal density with spatial separation and the evaluate differentiation potential of the surviving cells. MATERIALS AND METHODS: Pulp cells derived from wisdom teeth were seeded into wells of a 96-plate at a mean density of 1 cell/well and cultured for 2 weeks. Surviving cells were harvested, pooled together and subjected to differentiation into adipocytes, osteoblasts and neurons using respective inducing conditions for 3 weeks. The former two types of cells were examined by staining with Oil Red O and Alizarin Red, respectively. Neuron-like cells were inspected for their morphology and immunostained for microtubule-associated protein 2 and ß-tubulin III. RESULTS: Vital cells were obtained in eight wells of a 96-well plate, corresponding to a survival rate of 8%. Since fewer than two wells would be expected to contain more than four cells at seeding, the majority of surviving cells likely grew from 1-3 cells, which is a very low density. These cells differentiated into functional adipocytes and osteoblasts, and morphologically neuron-like cells. CONCLUSION: Low-density seeding with spatial separation enables statistical estimation of cell number in wells and provides an effective strategy for enriching stem/progenitor cells and for isolating clonal dental pulp cells.

The impact of collagen sponge composite bone marrow mesenchymal stem cells (BMSCs) in inducing interbody fusion.

OBJECTIVE: To assess the effectiveness of bone mesenchymal stem cells (BMSCs) in the induction of interbody fusion. PATIENTS AND METHODS: The 3rd generation BMSCs were seeded on collagen sponge scaffold and cultured in osteoblast induction medium for 3 weeks to prepare cell-scaffold complex. Thirty patients were randomly divided into three groups to establish the L4/L5 interbody fusion model. The cell-scaffold complex was implanted in the intervertebral space in group I, the collagen sponge scaffold was implanted in group II, and the autologous iliac crest spongy bone was implanted in group III. Palpation, radiography, micro-CT, and histology were performed on the 12th weeks after operation to evaluate osteogenesis and spinal fusion. RESULTS: BMSCs differentiated into osteoblasts in the cell-scaffold complex after osteogenic induction for 3 weeks. The spinal fusion rates in group I, II, and III were 40%, 0%, and 70%, respectively. Micro-CT and histological examination showed mature bone marrow and trabecular bone formation in the fusion segments. The new bone was integrated with the upper and lower vertebral body. The bone trabecula in group III was stronger than group I. The surgical segments in group II was scar tissue without collagen sponge residue. CONCLUSIONS: BMSCs can induce osseous fusion in the lumbar vertebra.

Effect of cell therapy with allogeneic osteoblasts on bone repair of rat calvaria defects.

BACKGROUND AIMS: Regenerative medicine strategies based on cell therapy are considered a promising approach to repair bone defects. The aims of this study were to evaluate the effect of subculturing on the osteogenic potential of osteoblasts derived from newborn rat calvaria and the effect of these osteoblasts on bone repair of rat calvaria defects. METHODS: Cells were obtained from 50 newborn rat calvaria, and primary osteoblasts (OB) were compared with first passage (OB-P1) in terms of osteogenic potential by assaying cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of the osteoblastic markers RUNX2, ALP, osteocalcin and bone sialoprotein. Then, 5-mm calvaria defects were created in 24 Wistar rats, and after 2 weeks, they were locally injected with 50 µL of phosphate-buffered saline containing either 5 × 106 osteoblasts (OB-P1, n = 12) or no cells (control, n = 12). Four weeks post-injection, the bone formation was evaluated by micro-computed tomography and histological analyses. Data were compared by analysis of variance, followed by the Student-Newman-Keuls's test or Student's t-test (P ≤ 0.05). RESULTS: OB-P1 showed high proliferation and ALP activity, and despite the reduced gene expression of osteoblastic markers and extracellular matrix mineralization compared with OB, they displayed osteogenic potential, being a good choice for injection into calvaria defects. The micro-tomographic and histological data showed that defects treated with OB-P1 presented higher bone formation compared with control defects. DISCUSSION: Our results indicate that cells derived from newborn rat calvaria retain osteoblastic characteristics after subculturing and that these osteoblasts stimulate bone repair in a rat calvaria defect model.

Porcine induced pluripotent stem cell-derived osteoblast-like cells prevent glucocorticoid-induced bone loss in Lanyu pigs.

The application of appropriate animal models and techniques for the study of osteoporosis is important. Lanyu pigs, a local miniature breed, have been widely used in various biomedical studies in Taiwan. This study aimed to induce bone loss in Lanyu pigs and to examine whether porcine induced pluripotent stem cell (piPSC)-derived osteoblast-like cells could recover bone mass of tibiae via local cell transplantation. piPSCs were directed to differentiate into osteoblast-like cells using osteogenic medium, and differentiated cells expressed osteogenic markers and phenotypes. Twenty mature female Lanyu pigs were divided into four groups, including control (C, 1% calcium diet), treatment 1 (T1, ovariectomy + 1% calcium diet), treatment 2 (T2, ovariectomy + 0.5% calcium diet), and treatment 3 (T3, ovariectomy + 0.5% calcium diet + 1 mg/kg of prednisolone) and were subjected to bone loss induction for twelve months. Micro-CT images revealed that the lowest trabecular bone parameters, such as trabecular bone volume, thickness, separation, number, and total porosity, were detected in the T3 group. The lowest proportions of cortical bone in the proximal metaphysis, proximal diaphysis, and distal diaphysis were also found in the T3 group. These results indicate that ovariectomy, calcium restriction, and prednisolone administration can be applied to induce proper bone loss in Lanyu pigs. After bone loss induction, pigs were subjected to cell transplantation in the left tibiae and were maintained for another six months. Results showed that transplanted piPSC-derived osteoblast-like cells significantly improved trabecular bone structures at transplanted sites and maintained cortical bone structures in the proximal metaphysis. In conclusion, the therapeutic potential of piPSC-derived osteoblast-like cells was confirmed via cell transplantation in the left tibiae of Lanyu pigs. These findings reveal the therapeutic potential of piPSCs for glucocorticoid-induced bone loss in pig models.

Alginate/poly(amidoamine) injectable hybrid hydrogel for cell delivery.

A covalently cross-linked injectable hybrid hydrogel, namely, alginate/poly(amidoamine) (PAMAM), with the objective of cell delivery was innovatively designed and synthesized using tetra-amino-functional PAMAM dendrimer as the cross-linker. With the increase in percentage of PAMAM cross-linker, the pore size and swelling ratio of hydrogels were in the range of 57 ± 18 µm to 88 ± 25 µm and 110 ± 16 to 157 ± 20, respectively. The study of attachment and proliferation of MC3T3-E1 pre-osteoblasts using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay through indirect and direct contact methods indicated a continuous increase in metabolically active live cells with time, implying non-cytotoxicity of the synthesized hydrogel. The live-dead assay showed >95% of live cells for alginate/PAMAM hydrogels, suggesting viability of the encapsulated cells. When the percentage of PAMAM cross-linker in alginate/PAMAM hydrogel was increased from 5 to 25, the percentage degradation rate decreased from 1.1 to 0.29%/day. Given that the poly(ethylene glycol) is commonly used cross-linker for hydrogel syntheses, we compared the behavior with poly(ethylene glycol). The incorporation of poly(ethylene glycol) in alginate/PAMAM hydrogel reduced the activity of MC3T3-E1 cells and their viability compared to the alginate/PAMAM hydrogels. The protonation of amino groups in alginate/PAMAM injectables under physiological conditions led to the formation of cationic hydrogels. These cationic hydrogels showed enhanced cell encapsulation and attachment ability because of electrostatic interaction with negatively charged cell surface as determined by cell adhesion and extensions from scanning electron microscope and vinculin assay and ability of in situ calcium phosphate mineralization. These observations point toward the potential use as an injectable scaffold for cell delivery and tissue engineering applications.

Peculiarities of Osteogenesis by Periosteal Cells after Experimental Ectopic Transplantation.

We carried out a comparative study of the features of osteogenesis from the progenitor osteogenic periosteal cells in rabbit and human. At the initial stages, high osteogenic potential of both human and rabbit periosteal cells was observed. However, at the later stages, the cell response favors resorption of the new bone tissue formed from periosteal cells in rabbits, but does not affect the bone tissue formed from human progenitor osteogenic periosteal cells. These functional characteristics of rabbit periosteal cells should be considered when planning the experiment.