Rabbit Bone Marrow Research Articles

Repopulation of multipotent stem cells derived from adult male rabbits on a polycaprolactone scaffold: an in vitro study

ABSTRACT Background: MSCs (mesenchymal stem cells) are multipotent, spindle-shaped cells that give them the ability to differentiate into a broad lineage of tissues. Polycaprolactone (PCL) is a synthetic material that has been used several times for the fabrication of scaffolds to be utilized in the field of tissue engineering. Aim of the work: The main goal of this research is to develop a protocol for isolating, cultivating, and characterizing rabbit bone marrow mesenchymal stem cells (BMSCs), as well as to investigate their capacity to repopulate PCL scaffolds. Results: In Dulbecco's Modified Eagle's Medium (DMEM) supplied with 10% fetal bovine serum (FBS), BMSCs were successfully isolated, cultivated, and grown. The cells were characterized morphologically by their spindle-shape, processes of cytoplasmic, plastic adhesion tendencies, and formation of colonies. They were characterized by flowcytometric analysis as having a positive CD73 expression and a negative CD45 expression. Before cell seeding, the PCL scaffold was inspected under a scanning electron microscope to assess the PCL material characteristics and pour size, and after cell seeding to detect BMSC attachments to the scaffold. Conclusion: The protocol used in this investigation was effective for isolation, culture, and characterization of rabbit BMSCs and the cells that were obtained successfully repopulated the PCL scaffold.

Hydroxyapatite-hybridized double-network hydrogel surface enhances differentiation of bone marrow-derived mesenchymal stem cells to osteogenic cells.

Recently, we have developed a hydroxyapatite (HAp)-hybridized double-network (DN) hydrogel (HAp/DN gel), which can robustly bond to the bone tissue in the living body. The purpose of this study is to clarify whether the HAp/DN gel surface can differentiate the bone marrow-derived mesenchymal stem cells (MSCs) to osteogenic cells. We used the MSCs which were harvested from the rabbit bone marrow and cultured on the polystyrene (PS) dish using the autogenous serum-supplemented medium. First, we confirmed the properties of MSCs by evaluating colony forming unit capacity, expression of MSC markers using flow cytometry, and multidifferential capacity. Secondly, polymerase chain reaction analysis demonstrated that the HAp/DN gel surface significantly enhanced mRNA expression of the eight osteogenic markers (TGF-β1, BMP-2, Runx2, Col-1, ALP, OPN, BSP, and OCN) in the cultured MSCs at 7 days than the PS surfaces (p < 0.0001), while the DN gel and HAp surfaces provided no or only a slight effect on the expression of these markers except for Runx2. Additionally, the alkaline phosphatase activity was significantly higher in the cells cultured on the HAp/DN gel surface than in the other three material surfaces (p < 0.0001). Thirdly, when the HAp/DN gel plug was implanted into the rabbit bone marrow, MSC marker-positive cells were recruited in the tissue generated around the plug at 3 days, and Runx2 and OCN were highly expressed in these cells. In conclusion, this study demonstrated that the HAp/DN gel surface can differentiate the MSCs into osteogenic cells.

Panax notoginseng Saponin Promotes Bone Regeneration in Distraction Osteogenesis via the TGF-β1 Signaling Pathway.

Distraction osteogenesis (DO) is an efficient strategy that is employed for the treatment of large bone defects in craniomaxillofacial surgery. Despite its utility, however, DO is associated with a prolonged consolidation phase and a high complication rate that hinder its more widespread utilization. Panax notoginseng saponin (PNS) is a traditional Chinese medicine that is frequently administered for the treatment of a range of conditions. Herein, we explored the ability of PNS treatment to influence osteogenic differentiation using both rabbit bone marrow mesenchymal cells (BMSCs) and a model of mandibular DO. BMSC proliferation was assessed via CCK-8 assay, while osteogenic differentiation was monitored through ALP and alizarin red S staining. A PCR approach was used to evaluate the expression of genes associated with osteogenesis (ALP, Runx2, and OCN) and genes linked to the TGF pathway (TβR-II, SMAD2, SMAD3, and PPM1A). For in vivo experiments, treated BMSCs were locally injected into the DO gap, with PNS being injected into treated rabbits every other day throughout the experimental period. The quality of the regenerative process was assessed via scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray imaging, and hematoxylin and eosin (H&E) staining. These analyses revealed that PNS was able to promote BMSC osteogenesis and mandibular generation, driving the upregulation of osteogenesis-related genes at the mRNA levels through the modulation of the TGF-β1/Smad pathway. Consistently, the overexpression or silencing of TβR-II in PNS-treated BMSCs was sufficient to modulate their osteogenic potential. Analyses of in vivo mandibular DO outcomes revealed significantly augmented new bone growth in the PNS-treated group relative to control animals, with maximal osteogenesis in the group overexpressing rabbit TβR-II. Together, these results highlight the PNS as a promising and cost-effective therapeutic tool with the potential to enhance bone regeneration in clinical contexts through the modulation of the TGF-β1/Smad pathway.

Magnesium-incorporated sol-gel Ta2O5 coating on Ti6Al4V and in vitro biocompatibility

Tantalum (Ta) has been used to modify the surface of titanium (Ti) due to its superior biological response under some clinically compromised situations such as osteoporosis and diabetes; however, the techniques used presented technical disadvantages. The currently study prepared Ta2O5 and Mg-incorporated Ta2O5 coatings on Ti6Al4V by a sol-gel method and compared in vitro response of rabbit bone marrow mesenchymal stem cells. Both coatings were submicrometer-thick and comprised nano-sized particles, the former being crystalline whereas the latter amorphous. Compared with uncoated Ti6Al4V, both coatings increased the corrosion resistance and water wettability, and improved the viability, proliferation, osteoblastic differentiation of the cells. Moreover, they also upregulated the expression of an angiogenesis-related gene (HIF-1α). With the exception of corrosion rate, greatest improvements were observed from the Mg-incorporated Ta2O5 coating. These improvements were probably due to the nanostructures and elements release of Mg and Ta from the coatings. All above results suggest that, Ta2O5-based coatings are promising tools for improving the biological response of Ti-based implants under compromised conditions.

Synergic adhesive chemistry-based fabrication of BMP-2 immobilized silk fibroin hydrogel functionalized with hybrid nanomaterial to augment osteogenic differentiation of rBMSCs for bone defect repair

Bone defect repair and tissue engineering is specifically challenging process because of the distinctive morphological and structural behaviours of natural bone with complex healing and biochemical mechanisms. In the present investigation, we designed dopamine adhesive chemistry-based fabrication of silk fibroin hydrogel (SFD) with incorporation of nano-hydroxyapatite (nHA)-graphene oxide (GO) hybrid nanofillers with well-arranged porous morphology immobilized with bone morphogenic protein-2 (BMP-2) for the effective in vitro rabbit bone marrow derived mesenchymal stem cells loading compatibility and in vivo new bone regrowth and collagen deposition ability. We have achieved bone-specific hydrogel scaffolds with upgraded structural features, mechanical properties and particularly promoted in vitro osteogenic differentiation and compatibility of rabbit bone marrow mesenchymal stem cells (rBMSCs). Structural and microscopic analyses established greater distributions of components and well-ordered and aligned porous structure of the hydrogel network. In vivo result of new bone regrowth was promisingly higher in the Bm@nHG-SFD hydrogel (85%) group as compared to the other treatment groups of nHG-SFD (77%) and nH-SFD (64%) hydrogel. Overall, we summarized that morphologically improved hydrogel material with immobilization of BMP-2 could be have more attentions for new generation bone regeneration therapies.

Regeneration of Bone Defects in a Rabbit Femoral Osteonecrosis Model Using 3D-Printed Poly (Epsilon-Caprolactone)/Nanoparticulate Willemite Composite Scaffolds.

Steroid-associated osteonecrosis (SAON) is a chronic disease that leads to the destruction and collapse of bone near the joint that is subjected to weight bearing, ultimately resulting in a loss of hip and knee function. Zn2+ ions, as an essential trace element, have functional roles in improving the immunophysiological cellular environment, accelerating bone regeneration, and inhibiting biofilm formation. In this study, we reconstruct SAON lesions with a three-dimensional (3D)-a printed composite made of poly (epsilon-caprolactone) (PCL) and nanoparticulate Willemite (npW). Rabbit bone marrow stem cells were used to evaluate the cytocompatibility and osteogenic differentiation capability of the PCL/npW composite scaffolds. The 2-month bone regeneration was assessed by a Micro-computed tomography (micro-CT) scan and the expression of bone regeneration proteins by Western blot. Compared with the neat PCL group, PCL/npW scaffolds exhibited significantly increased cytocompatibility and osteogenic activity. This finding reveals a new concept for the design of a 3D-printed PCL/npW composite-based bone substitute for the early treatment of osteonecrosis defects.

Fabrication of Injectable Chitosan-Chondroitin Sulfate Hydrogel Embedding Kartogenin-Loaded Microspheres as an Ultrasound-Triggered Drug Delivery System for Cartilage Tissue Engineering.

Ultrasound-responsive microspheres (MPs) derived from natural polysaccharides and injectable hydrogels have been widely investigated as a biocompatible, biodegradable, and controllable drug delivery system and cell scaffolds for tissue engineering. In this study, kartogenin (KGN) loaded poly (lactide-co-glycolic acid) (PLGA) MPs (MPs@KGN) were fabricated by premix membrane emulsification (PME) method which were sonicated by an ultrasound transducer. Furthermore, carboxymethyl chitosan-oxidized chondroitin sulfate (CMC-OCS) hydrogel were prepared via the Schiff’ base reaction-embedded MPs to produce a CMC-OCS/MPs scaffold. In the current work, morphology, mechanical property, porosity determination, swelling property, in vitro degradation, KGN release from scaffolds, cytotoxicity, and cell bioactivity were investigated. The results showed that MPs presented an obvious collapse after ultrasound treatment. The embedded PLGA MPs could enhance the compressive elastic modulus of soft CMC-OCS hydrogel. The cumulative release KGN from MPs exhibited a slow rate which would display an appropriate collapse after ultrasound, allowing KGN to maintain a continuous concentration for at least 28 days. Moreover, the composite CMC-OCS@MPs scaffolds exhibited faster gelation, lower swelling ratio, and lower in vitro degradation. CCK-8 and LIVE/DEAD staining showed these scaffolds did not influence rabbit bone marrow mesenchymal stem cells (rBMMSCs) proliferation. Then these scaffolds were cultured with rBMMSCs for 2 weeks, and the immunofluorescent staining of collagen II (COL-2) showed that CMC-OCS hydrogel embedded with MPs@KGN (CMC-OCS@MPs@KGN) with ultrasound had the ability to increase the COL-2 synthesis. Overall, due to the improved mechanical property and the ability of sustained KGN release, this injectable hydrogel with ultrasound-responsive property is a promising system for cartilage tissue engineering.

Effect of Activated Platelet-Rich Plasma on Chondrogenic Differentiation of Rabbit Bone Marrow-Derived Mesenchymal Stem Cells

We aimed to evaluate the effect of activated platelet-rich plasma (PRP) on proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Six mature male rabbits were included in this study. PRP was obtained by two-step centrifugation from whole blood, and it was activated using CaCl2 solution. BMSCs were isolated and proliferated from bone marrow of rabbits and characterized by flow cytometry. Passage 3 BMSCs were cultured in high-glucose Dulbecco's modified Eagle's medium (HG-DMEM) with the four different compositions for consecutive 7 days, including 10% fetal bovine serum, 5% PRP, 10% PRP, and 15% PRP. Cell counting assays were performed to evaluate the cell proliferation of BMSCs. BMSCs (5 × 105 cells/well in 6-well plates) were induced in four conditions for 21 days to chondrogenic differentiation evaluation, including commercial chondrogenic medium (control), 5% PRP (HG-DMEM+5% PRP), 10% PRP (HG-DMEM+10% PRP), and 15% PRP (HG-DMEM+15% PRP). The gene expression levels of ACAN, COL2A1, and SOX9 in pellets were detected. Morphological and pathological assessments were performed by the blind observer. After purifying, the percentages of cells with CD105(+)/CD34(−) and CD44(+)/CD45(−) were 96.5% and 92.9%, respectively. The proliferation of BMSCs was enhanced in all groups, and 10% PRP revealed more significant outcome than the others from day 5. The levels of ACAN, COL2A1, and SOX9 were lower in the three PRP groups than control group, but the levels of ACAN and SOX9 were higher in 10% PRP group than 5% and 15% PRP groups. Histological examinations showed that 10% PRP-treated pellets had more regular appearance, larger size, and abundant extracellular matrix than 5% or 10% PRP groups, but still inferior to commercial chondrogenic medium. In conclusion, our results show that PRP may enhance the proliferation of rabbit BMSCs. However, PRP have limited effect on chondrogenic differentiation in comparison with commercial chondrogenic medium in pellets culture.

Autophagy stimulation delayed biological aging and decreased cardiac differentiation in rabbit mesenchymal stem cells.

Introduction: Cardiovascular disease (CVD) is a type of disease that affects the function of cardiac-vascular tissues. This study aimed to consider the possible effects of autophagy, as an intrinsic catabolic pathway of cells, on the differentiation and aging process of mesenchymal stem cells (MSCs). Methods: In this study, bone marrow-derived MSCs were obtained from rabbit bone marrow aspirates. The stemness feature was confirmed by using flow cytometry analysis Cells at passage three were treated with 50 μM Metformin and 15μM hydroxychloroquine (HCQ) for 72 hours. The intracellular accumulation of autophagolysosomes was imaged using LysoTracker staining. Protein levels of autophagy (LC3II/I ratio), aging (Klotho, PARP-1, and Sirt-1) effectors, and cardiomyocyte-like phenotype (α-actinin) were studied by western blotting. Results: Based on our findings, flow cytometry analysis showed that the obtained cells expressed CD44 and CD133 strongly, and CD31 and CD34 dimly, showing a typical characteristic of MSCs. Our data confirmed an increased LC3II/I ratio in the metformin-received group compared to the untreated and HCQ-treated cells (P < 0.05). Besides, we showed that the incubation of rabbit MSCs with HCQ increased cellular aging by induction of PARP-1 while Metformin increased rejuvenating factor Sirt-1 comparing with the normal group (P < 0.05). Western blotting data showed that the autophagy stimulation response in rabbit MSCs postponed the biological aging and decreased the differentiation potential to the cardiac cells by diminishing α-actinin comparing with control cells (P < 0.05). Conclusion: In summary, for the informants in this study, it could be noted that autophagy inhibition/stimulation could alter rabbit MSCs aging and differentiation capacity.

Construction and identification of recombinant adenovirus vector and observation of its transfecting rabbit bone marrow mesenchymal stem cells

To construct and identify adenovirus vector co-expressing hBMP2 and hVEGF165 fusion protein which labeled with green fluorescence protein, and laying the foundtion of the effect of hBMP2 and hVEGF165 gene inducing BMMSCs differentiation to osteoblast and bone defect repaired in the body. BMP2 and VEGF165 gene was amplified from cDNA library by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. Ad-BMP2- VEGF165 was recombinated and propagated in HEK293 cells by co-transfecting with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ E1, 3Cre. The recombinant adenovirus was purified and virustiter was determined, and then to research GFP expression and to calculate the adenovirus transfection rate in rabbit BMMSCs. The recombinant adenovirus vector Ad-BMP2-VEGF165 was successfully constructed by the methods of gene analyzing, colony PCR, Western blotting and observing GFP expression, and the titer of the adenovirus was 1×1010 PFU /ml. Recombinant adenovirus vector containing hBMP2 and hVEGF165 gene was successfully constructed and its high titer was obtained.

SSEA-4 Antigen Is Expressed on Rabbit Lymphocyte Subsets

SSEA-4 antigen can be mainly found in embryos and embryonic stem cells. However, its expression has been observed also in adult stem and progenitor cells, or even in some differentiated cells. Moreover, we found a considerable number of SSEA-4 positive (SSEA-4+) cells within the rabbit peripheral blood and bone marrow mononuclear cells (PBMCs and BMMCs) in our previous study. Since no information about such cells can be found anywhere in the literature, the aim of this study was to identify their origin. At first, phenotypic analyses of fresh rabbit PBMCs and BMMCs were performed using flow cytometry and specific antibodies against SSEA-4 and leukocyte subsets. Then, SSEA-4+ were enriched using magnetic activated cell sorting (MACS) and analyzed for their phenotype using qPCR. We found significant SSEA-4+ cell population in PBMCs (~50%) and BMMCs (~20%). All those cells co-expressed CD45 and a majority of them also expressed B-cell marker (IgM; 50% of SSEA-4+ PBMCs and 60% of SSEA-4+ BMMCs). Increased (p &lt; 0.05) expression of SSEA-4, CD45 and B-cell markers (IgM, CD79α and MHCII) were also noticed by qPCR in SSEA-4+ cells enriched via MACS (with efficiency over 80%). Both methods did not detect significant expression of monocyte or T-cell markers. In conclusion, SSEA-4+ cells in rabbit blood and bone marrow are of hematopoietic origin and probably belong to B-lineage cells as possessing the phenotype of B lymphocytes. However, the true function of SSEA-4 antigen in these cells should be explored by further studies.

Bioinspired polysaccharide hybrid hydrogel promoted recruitment and chondrogenic differentiation of bone marrow mesenchymal stem cells

Cartilage regeneration by biomimetic cartilage matrix with synchronously recruited stem cells was one of ideal strategies. Inspired by catechol for proteins adhesion, dopamine modified polysaccharide hybrid hydrogel (HD-C) was prepared by integrating collagen I (Col I) and hyaluronic acid derivatives (HA-DN) with sulfhydryl modified polysaccharide hybrid hydrogel (HS-C) as control. Because of double-crosslinking architecture, HD-C hydrogel was endowed with a more compact pore structure, higher mechanical properties and water retention ability in comparison with those of HS-C hydrogel. Meanwhile, it significantly promoted the proliferation and spread of rabbit bone marrow stem cells (rBMSCs), and accelerated cartilaginous matrix secretion. RT-PCR results also verified higher related gene expression of chondrogenesis (Sox 9, Agg and Col II). Moreover, HD-C hydrogel could enhance the enrichment and migration of rBMSCs in vitro by potential functional protein adsorption mechanisms, and this phenomenon was further confirmed by more rBMSCs migration in short-term joint implantation experiments in vivo.

Culture of rabbit bone marrow mesenchymal stem cells on polyurethane/pyrrole surface promoted differentiation into endothelial lineage.

Due to the electrical conductivity, pyrrole-based scaffolds are one of the attractive biomaterials in the regeneration of electrically active tissues like the heart and brain. Here, we investigated the impact of polyurethane/pyrrole scaffold on the angiogenesis differentiation of rabbit mesenchymal stem cells toward endothelial lineage in vitro. Nanoelectrospun polyurethane/pyrrole fibers were synthesized and characterized using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrum analysis, scanning electron microscope (SEM) imaging. Mechanical properties, electroconductivity, and hydrophobicity were also measured. The viability of cells was monitored 72hours after being plated on the polyurethane/pyrrole surface. The endothelial differentiation of stem cells was explored using western blotting. ATR-FTIR revealed that the pyrrole was successfully polymerized to polypyrrole and blend with polyurethane fibers. The addition of pyrrole to polyurethane increased the tensile strength compared to the polyurethane group. These features coincided with the reduction of the hydrophilic properties of polyurethane. Based on our data, the electro-conductivity of polyurethane/pyrrole was superior compared to the polyurethane group. SEM imaging showed an appropriate cell attachment to the surface of polyurethane/pyrrole and polyurethane groups synthesized membranes. MTT assay revealed a significantly increased survival rate in the polyurethane/pyrrole group compared to the polyurethane group (P<.05). We noted a statistically significant increase of endothelial-associated proteins, CD31, von Willebrand factor, and CD34, in cells expanded on polyurethane/pyrrole compared to the polyurethane group (P<.05). As a more general note, it could be hypothesized that the polyurethane/pyrrole blend could improve the angiogenesis potency of rabbit bone marrow mesenchymal stem cells for regenerative purposes.

Effect of a plasma synthesized polypyrrole coverage on polylactic acid/hydroxyapatite scaffolds for bone tissue engineering.

Composite biomaterials are solids that contain two or more different materials, combining the properties of their components to restore or improve the function of tissues. In this study, we report the generation of electrospun matrices with osteoconductive properties and porosity using the combination of a biodegradable polyester, polylactic acid (PLA), and hydroxyapatite (HA). Additionally, we report the effects of modifying these matrices through plasma polymerization of pyrrole on the growth and osteogenic differentiation of rabbit bone marrow stem cells. Cells were isolated, seeded and cultured on biomaterials for periods between 7 and 28 days. The matrices we obtained were formed by nano and microfibers containing up to 35.7wt% HA, presenting a variety of apparent pore sizes to allow for the passage of nutrients to bone cells. Scanning electron microscopy showed that the fibers were coated with polypyrrole doped with iodine, and MTT assay demonstrated this increased cell proliferation and significantly improved cell viability due to the adhesive properties of the polymer. Our results show that PLA/HA/Pyrrole/Iodine matrices are favorable for bone tissue engineering.

Bone Mesenchymal Stem Cells Contribute to Ligament Regeneration and Graft-Bone Healing after Anterior Cruciate Ligament Reconstruction with Silk-Collagen Scaffold.

Anterior cruciate ligament (ACL) reconstruction was realized using a combination of bone mesenchymal stem cells (BMSCs) and silk–collagen scaffold, and an in vivo evaluation of this combination was performed. By combining type I collagen and degummed silk fibroin mesh, silk–collagen scaffolds were prepared to simulate ligament components. BMSCs isolated from bone marrow of rabbits were cultured for a homogenous population and seeded on the silk–collagen scaffold. In the scaffold and BMSC (S/C) group, scaffolds were seeded with BMSCs for 72 h and then rolled and used to replace the ACL in 20 rabbits. In the scaffold (S) group, scaffolds immersed only in culture medium for 72 h were used for ACL reconstruction. Specimens were collected at 4 and 16 weeks postoperatively to assess ligament regeneration and bone integration. HE and immunohistochemical staining (IHC) were performed to assess ligament regeneration in the knee cavity. To assess bone integration at the graft–bone interface, HE, Russell–Movat staining, micro-CT, and biomechanical tests were performed. After 4 weeks, vigorous cell proliferation was observed in the core part of the scaffold in the S/C group, and a quantity of fibroblast-like cells and extracellular matrix (ECM) was observed in the center part of the graft at 16 weeks after surgery. At 4 and 16 weeks postoperatively, the tenascin-C expression in the S/C group was considerably higher than that in the S group (4 w, p < 0.01; 16 w, p < 0.01). Furthermore, bone integration was better in the S/C group than in the S group, with histological observation of trabecular bone growth into the graft and more mineralized tissue formation detected by micro-CT (4 w, bone volume fraction (BV/TV), p = 0.0169, bone mineral density (BMD), p = 0.0001; 16 w, BV/TV, p = 0.1233, BMD, p = 0.0494). These results indicate that BMSCs promote ligament regeneration in the knee cavity and bone integration at the graft–bone interface. Silk–collagen scaffolds and BMSCs will likely be combined for clinical practice in the future.

Corneal reconstruction in chemically damaged cornea using temperature responsive surface assisted mesenchymal stem cell transplantation in rabbits.

Transplantation of autologous stem cells over damaged cornea seems to be a promising approach for corneal reconstruction. Use of a biocompatible carrier is still a challenge in bedside translation of transplantation. We investigated corneal reconstruction and tissue remodelling by transplantation of mesenchymal stem cells (MSCs) using temperature responsive membranes in chemically damaged rabbit cornea model. MSCs were cultured from rabbit's bone marrow and transplanted over alkali injured cornea, using either temperature responsive membrane or fibrin glue method. Endogenous levels of MSCs were assessed to decide the optimal time point for transplanting cells. MSC transplanted corneas were harvested at different time points post-transplantation. Corneal repair markers were evaluated using histopathology, immunohistochemistry (IHC) and real time qPCR. The quality of cornea reconstructed was evaluated and compared using corneal opacity scoring and immunohistochemistry (IHC). Use of temperature responsive surface as carrier resulted in uniform and homogenous delivery of MSCs sheet over the damaged corneal surface. Corneal transparency improved day 7 onwards post-MSC transplantation in rabbit chemically injured cornea. Complete re-epithelialization of injured cornea was observed 15 days after MSC transplantation. Restoration of vimentin, α-smooth muscle actin and collagen levels in MSC transplanted cornea was observed post-transplantation. Further, differentiation of MSCs into mature corneal epithelial cells was also observed upon transplantation. The extent of corneal repair was apparently better using temperature responsive surfaces. The surface provides biocompatible niche for MSCs and can be a method of choice in clinics for cell transplantation over the damaged ocular surfaces.

The Application of a Bone Marrow Mesenchymal Stem Cell Membrane in the Vascularization of a Decellularized Tracheal Scaffold.

Airway stenosis is a common problem in the neonatal intensive care unit (NICU) and pediatric intensive care unit (PICU). A tissue-engineered trachea is a new therapeutic method and a research hotspot. Successful vascularization is the key to the application of a tissue-engineered trachea. However, successful vascularization studies lack a complete description. In this study, it was assumed that rabbit bone marrow mesenchymal stem cells were obtained and induced by ascorbic acid to detect the tissue structure, ultrastructure, and gene expression of the extracellular matrix. A vascular endothelial cell culture medium was added in vitro to induce the vascularization of the stem cell sheet (SCS), and the immunohistochemistry and gene expression of vascular endothelial cell markers were detected. At the same time, vascular growth-related factors were added and detected during SCS construction. After the SCS and decellularized tracheal (DT) were constructed, a tetrandrine allograft was performed to observe its vascularization potential. We established the architecture and identified rabbit bone marrow mesenchymal stem cell membranes by 14 days of ascorbic acid, studied the role of a vascularized membrane in inducing bone marrow mesenchymal stem cells by in vitro ascorbic acid, and assessed the role of combining the stem cell membranes and noncellular tracheal scaffolds in vivo. Fourteen experiments confirmed that cell membranes promote angiogenesis at gene level. The results of 21-day in vitro experiments showed that the composite tissue-engineered trachea had strong angiogenesis. In vivo experiments show that a composite tissue-engineered trachea has strong potential for angiogenesis. It promotes the understanding of diseases of airway stenosis and tissue-engineered tracheal regeneration in newborns and small infants.

Shapable bulk agarose–gelatine–hydroxyapatite–minocycline nanocomposite fabricated using a mineralizing system aided with electrophoresis for bone tissue regeneration

To develop a shapable bulk antibacterial nanocomposite biomaterial for bone regeneration. A bulk agarose–gelatine hydrogel was mineralized using a hydrogel mineralizing system aided with electrophoresis, and the mineralized hydrogel was loaded with minocycline to obtain the agarose–gelatine–hydroxyapatite–minocycline nanocomposite. The nanocomposite had a large Brunauer–Emmett–Teller surface area of 44.4518 m2 g−1 and a high porosity of 76.9%. Hydroxyapatite crystals were well developed in the hydrogel matrix and exhibited a hybrid structure of microscale and nanoscale motifs. The addition of minocycline resulted in a continuous antibiotic release, inhibiting the growth of Staphylococcus aureus over 2 weeks in vitro. Exposed to rabbit bone marrow mesenchymal stem cells, the nanocomposite revealed good cytocompatibility in vitro. Furthermore, the biomaterial could effectively enhance the bone regeneration in a critical-size rabbit cranial defect model in vivo. These findings depicted that the nanocomposite, with good biocompatibility and good antibacterial property, is a promising candidate for future clinical application in bone tissue engineering or as a prospective bone replacement biomaterial.

Transplantation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Improves Cartilage Repair in a Rabbit Model.

The aim of this study was to investigate the therapeutic efficacy and safety of transplanting human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the treatment of cartilage injury. First, the articular cartilage defect model in rabbits was constructed. Then, the identified hUCB-MSCs and rabbit bone marrow stem cells (rBM-MSCs) were transplanted into the bone defect, respectively, and the cartilage repair effect was observed by hematoxylin-eosin (HE) staining and immunohistochemistry. Besides, the glycosaminoglycan (GAG) content and biomechanics of the restoration area were also evaluated. In our study, hUCB-MSCs and rBM-MSCs exhibited typical MSC characteristics, with positive expressions of CD73, CD105, and CD90 and negative for CD45, CD34, CD14, and HLA-DR. After the transplantation of hUCB-MSCs and rBM-MSCs, the overall quality of cartilage tissue was significantly improved, and the recipients did not show significant side effects in general. However, the expression of matrix metalloproteinase-13 (MMP-13) in the de novo tissues of the hUCB-MSCs and rBM-MSCs groups was both increased, indicating that the novel tissues may have some potential osteoarthritic changes. In conclusion, our results suggest the therapeutic effect of hUCB-MSCs transplantation in cartilage regeneration, providing a promising future in the clinical treatment of cartilage injury.

Enrichment of Rabbit Primitive Hematopoietic Cells via MACS Depletion of CD45+ Bone Marrow Cells

Hematopoietic stem and progenitor cells (HSC/HPCs) of human or few animal species have been studied for over 30 years. However, there is no information about rabbit HSC/HPCs, although they might be a valuable animal model for studying human hematopoietic disorders or could serve as genetic resource for the preservation of animal biodiversity. CD34 marker is commonly used to isolate HSC/HPCs. Due to unavailability of specific anti-rabbit CD34 antibodies, a novel strategy for the isolation and enrichment of rabbit HSC/HPCs was used in this study. Briefly, rabbit bone marrow mononuclear cells (BMMCs) were sorted immunomagnetically in order to remove all mature (CD45+) cells. The cells were depleted with overall purity about 60–70% and then cultured in a special medium designed for the expansion of CD34+ cells. Quantitative Polymerase Chain Reaction (qPCR) analysis confirmed the enrichment of primitive hematopoietic cells, as the expression of CD34 and CD49f increased (p &lt; 0.05) and CD45 decreased (p &lt; 0.001) at the end of culture in comparison to fresh BMMCs. However, cell culture still exhibited the presence of CD45+ cells, as identified by flow cytometry. After gating on CD45− cells the MHCI+MHCII−CD38+CD49f+CD90−CD117− phenotype was observed. In conclusion, rabbit HSC/HPCs might be isolated and enriched by the presented method. However, further optimization is still required.