Rabbit Bone Marrow Stem Cells Research Articles

3-Dimensional Bioprinting of a Tendon Stem Cell–Derived Exosomes Loaded Scaffold to Bridge the Unrepairable Massive Rotator Cuff Tear

Background: Unrepairable massive rotator cuff tears (UMRCTs) are challenging to surgeons owing to the severely retracted rotator cuff musculotendinous tissues and extreme defects in the rotator cuff tendinous tissues. Purpose: To fabricate a tendon stem cell–derived exosomes loaded scaffold (TSC-Exos-S) and investigate its effects on cellular bioactivity in vitro and repair in a rabbit UMRCT model in vivo. Study Design: Controlled laboratory study. Methods: TSC-Exos-S was fabricated by loading TSC-Exos and type 1 collagen (COL-I) into a 3-dimensional bioprinted and polycaprolactone (PCL)–based scaffold. The proliferation, migration, and tenogenic differentiation activities of rabbit bone marrow stem cells (BMSCs) were evaluated in vitro by culturing them in saline, PCL-based scaffold (S), COL-I loaded scaffold (COL-I-S), and TSC-Exos-S. In vivo studies were conducted on a rabbit UMRCT model, where bridging was repaired with S, COL-I-S, TSC-Exos-S, and autologous fascia lata (FL). Histological and biomechanical analyses were performed at 8 and 16 weeks postoperatively. Results: TSC-Exos-S exhibited reliable mechanical strength and subcutaneous degradation, which did not occur before tissue regeneration. TSC-Exos-S significantly promoted the proliferation, migration, and tenogenic differentiation of rabbit BMSCs in vitro. In vivo studies showed that UMRCT repaired with TSC-Exos-S exhibited significant signs of tendinous tissue regeneration at the bridging site with regard to specific collagen staining. Moreover, no significant differences were observed in the histological and biomechanical properties compared with those repaired with autologous FL. Conclusion: TSC-Exos-S achieved tendinous tissue regeneration in UMRCT by providing mechanical support and promoting the trend toward tenogenic differentiation. Clinical Relevance: The present study proposes a potential strategy for repairing UMRCT with severely retracted musculotendinous tissues and large tendinous tissue defects.

Potentiostatic preparation and in vitro characterization of functional hazenite conversion coatings on AZ31 magnesium alloy

A functional hazenite coating was deposited on AZ31 Mg alloy by polarization at −0.8 V in a deaerated phosphate solution containing 0.1 M K2HPO4 and 0.1 M Na2HPO4 with pH 9. The growth process of the coating was determined by analyzing its morphological and composition evolution. The roughness, hydrophilicity, long-term protectiveness, bioactivity and bio-compatibility of the coating were characterized. The stress corrosion cracking (SCC) susceptibility of the coated AZ31 Mg alloy was also investigated. The results showed that the coating consisted of crystallized Mg(OH)2, MgHPO4·3H2O, KMgPO4·6H2O and KNaMg2(PO4)2·14H2O. The latter three compounds were transformed into hydroxyapatite, improving the bioactivity of AZ31 Mg alloy. The coating increased the roughness and hydrophilicity and significantly stimulated the proliferation of rabbit bone marrow stem cells, enhancing the biocompatibility of AZ31 Mg alloy. Additionally, the coating reduced the degradation rate of AZ31 Mg alloy by more than two orders of magnitude and the hydrogen volume by 50.9 % and showed good long-term protectiveness. Moreover, the coating decreased the SCC susceptibility indexes IUTS and Iε of AZ31 Mg alloy by 41.9 % and 44.9 %, respectively.

Microstructurally and mechanically tunable acellular hydrogel scaffold using carboxymethyl cellulose for potential osteochondral tissue engineering

In tissue engineering, scaffold microstructures and mechanical cues play a significant role in regulating stem cell differentiation, proliferation, and infiltration, offering a promising strategy for osteochondral tissue repair. In this present study, we aimed to develop a facile method to fabricate an acellular hydrogel scaffold (AHS) with tunable mechanical stiffness and microstructures using carboxymethyl cellulose (CMC). The impacts of the degree of crosslinking, crosslinker length, and matrix density on the AHS were investigated using different characterization methods, and the in vitro biocompatible of AHS was also examined. Our CMC-based AHS showed tunable mechanical stiffness ranging from 50 kPa to 300 kPa and adjustable microporous size between 50 μm and 200 μm. In addition, the AHS was also proven biocompatible and did not negatively affect rabbit bone marrow stem cells' dual-linage differentiation into osteoblasts and chondrocytes. In conclusion, our approach may present a promising method in osteochondral tissue engineering.

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.

MicroRNA (miR)-29 Modified Bone Marrow Stem Cells (BMSCs) Regulating Wnt-β-Catenin-p53 Pathway to Inhibit the Migration and Proliferation of Conjunctival Fibroblasts

To explore the mechanism of miR-29 modified bone marrow stem cells (BMSCs) in regulating the Wnt/β-Catenin-p53 pathway to inhibit the migration and proliferation of conjunctival fibroblasts. New Zealand rabbit BMSCs were randomly divided into control group, miR-29NC group, and miR-29mimic group. MTT assay, cell scratch test, and Western blot were used to detect the proliferation, migration, and protein expressions in constituent fibroblast. Cell proliferation in the miR-29mimic group was attenuated at 24 h and 48 h (P <0.05). The expression of miR-29 in miR-29 mimic group was upregulated, while Wnt/β-Catenin-p53 protein was decreased (P < 0.05). MiR-29 modified BMSC can inhibit the expression of Wnt/β-Catenin-p53 pathway and suppress the proliferation and migration of conjunctival fibroblasts.

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.

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.

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.

Fabrication and Characterization of Silk Fibroin Microfiber-Incorporated Bone Marrow Stem Cell Spheroids to Promote Cell-Cell Interaction and Osteogenesis.

In this study, silk fibroin microfiber (mSF) was applied to assist spheroid assemblies of rBMSCs (rabbit bone marrow stem cells) (S/B). Alkaline hydrolysis was induced with different times and conditions to manufacture the various sizes of mSF. The mSF was incorporated in the rBMSC with different amounts to optimize proper content for spheroid assembly. The formation of the S/B was confirmed under optical microscopy and SEM. Proliferation and viability were characterized by CCK-8 and live/dead staining. Osteogenesis was analyzed with ALP (alkaline phosphatase) activity studies and real-time polymerase chain reaction. The S/B was successfully produced and displayed uniformly distributed cells and mSF with the presence of a gap in the structure. Proliferation and viability of the S/B significantly increased when compared to rBMSC spheroids (B), which is potentially due to the enhanced transportation of oxygen and nutrients to the cells located in the core region. Additionally, ALP activity and osteogenic markers were significantly upregulated in the optimized S/B under osteogenic media conditions. Overall, a hybrid–spheroid system with a simple 3D cell culture platform provides a potential approach for engineering therapeutic stem cells.

Preparation and Characterization of Surface Heat Sintered Nanohydroxyapatite and Nanowhitlockite Embedded Poly (Lactic-co-glycolic Acid) Microsphere Bone Graft Scaffolds: In Vitro and in Vivo Studies.

In the context of using bone graft materials to restore and improve the function of damaged bone tissues, macroporous biodegradable composite bone graft scaffolds have osteoinductive properties that allow them to provide a suitable environment for bone regeneration. Hydroxyapatite (HAP) and whitlockite (WLKT) are the two major components of hard tissues such as bone and teeth. Because of their biocompatibility and osteoinductivity, we synthesized HAP (nHAP) and WLKT nanoparticles (nWLKT) by using the chemical precipitation method. The nanoparticles were separately incorporated within poly (lactic-co-glycolic acid) (PLGA) microspheres. Following this, the composite microspheres were converted to macroporous bone grafts with sufficient mechanical strength in pin or screw shape through surface sintering. We characterized physico-chemical and mechanical properties of the nanoparticles and composites. The biocompatibility of the grafts was further tested through in vitro cell adhesion and proliferation studies using rabbit bone marrow stem cells. The ability to promote osteogenic differentiation was tested through alkaline phosphate activity and immunofluorescence staining of bone marker proteins. For in vivo study, the bone pins were implanted in tibia bone defects in rabbits to compare the bone regeneration ability though H&E, Masson’s trichrome and immunohistochemical staining. The results revealed similar physico-chemical characteristics and cellular response of PLGA/nHAP and PLGA/nWLKT scaffolds but the latter is associated with higher osteogenic potential towards BMSCs, pointing out the possibility to use this ceramic nanoparticle to prepare a sintered composite microsphere scaffold for potential bone grafts and tissue engineered implants.

A novel knitted scaffold made of microfiber/nanofiber core-sheath yarns for tendon tissue engineering.

Tendon injury is common in sports and other rigorous activities, which may result in dysfunction and disability. Recently, scaffolds with a knitted structure have been widely applied for tendon tissue engineering. The purpose of this study was to fabricate a novel knitted tendon scaffold made of microfiber/nanofiber core-sheath yarns and evaluate the biocompatibility and the effect of tenogenic differentiation and tendon tissue regeneration in vitro and in vivo. Poly(ε-caprolactone) (PCL) microfibers, PCL microfibers-PCL nanofibers (PCL-PCL) and PCL microfiber-silk fibroin/poly(l-lactic acid-co-ε-caprolactone) nanofiber (SF/PLCL) core-sheath yarns were fabricated and then knitted with an automatic knitting machine to produce PCL, PCL-PCL and PCL-SF/PLCL fabric scaffolds. The characterization of the scaffolds was performed by using scanning electron microscopy, attenuated total reflectance Fourier transform infrared spectroscopy and an universal mechanical instrument. The in vitro experiment showed that rabbit bone marrow stem cells seeded on the scaffolds exhibited an elongated morphology and proliferated better in the PCL-SF/PLCL group, as compared to the PCL and PCL-PCL groups. Moreover, the PCL-SF/PLCL scaffold promoted the tenogenic differentiation of the cells for the highest expression levels of the tendon-related genes through down-regulating p-ERK1/2 expression among the three groups. Furthermore, the in vivo study in a rabbit patellar defect model demonstrated that the PCL-SF/PLCL scaffold could enhance the tissue regeneration and remodeling process as indicated by the better structural and biomechanical properties according to the results of histology, immunohistochemistry, transmission electron microscope examination and biomechanical tests. Therefore, the PCL-SF/PLCL scaffold is proved to be a promising biomaterial for tendon tissue engineering and a potential candidate for clinical treatment of tendon injury in the future.

Effect of strontium-containing on the properties of Mg-doped wollastonite bioceramic scaffolds

BackgroundBone scaffold is one of the most effective methods to treat bone defect. The ideal scaffold of bone tissue should not only provide space for bone tissue growth, but also have sufficient mechanical strength to support the bone defect area. Moreover, the scaffold should provide a customized size or shape for the patient’s bone defect.MethodsIn this study, strontium-containing Mg-doped wollastonite (Sr-CSM) bioceramic scaffolds with controllable pore size and pore structure were manufactured by direct ink writing 3D printing. Biological properties of Sr-CSM scaffolds were evaluated by apatite formation ability, in vitro proliferation ability of rabbit bone-marrow stem cells (rBMSCs), and alkaline phosphatase (ALP) activity using β-TCP and Mg-doped wollastonite (CSM) scaffolds as control. The compression strength of three scaffold specimens was probed after completely drying them while been submerged in Tris–HCl solution for 0, 2,4 and 6 weeks.ResultsThe mechanical test results showed that strontium-containing Mg-doped wollastonite (Sr-CSM) scaffolds had acceptable initial compression strength (56 MPa) and maintained good mechanical stability during degradation in vitro. Biological experiments showed that Sr-CSM scaffolds had a better apatite formation ability. Cell experiments showed that Sr-CSM scaffold had a higher cell proliferation ability compared with β-TCP and CSM scaffold. The higher ALP activity of Sr-CSM scaffold indicates that it can better stimulate osteoblastic differentiation and bone mineralization.ConclusionsTherefore, Sr-CSM scaffolds not only have acceptable compression strength, but also have higher osteogenesis bioactivity, which can be used in bone tissue engineering scaffolds.

Sonic hedgehog promotes chondrogenesis of rabbit bone marrow stem cells in a rotary cell culture system

BackgroundSonic hedgehog (Shh) is an important signalling protein involved in the induction of early cartilaginous differentiation. Herein, we demonstrate that Shh markedly induces chondrogenesis of rabbit bone marrow stromal cells (BMSCs) under microgravity conditions, and promotes cartilage regeneration.ResultsIn the rotary cell culture system (RCCS), chondrogenic differentiation was revealed by stronger Toluidine Blue and collagen II immunohistochemical staining in the Shh transfection group, and chondroinductive activity of Shh was equivalent to that of TGF-β. Western blotting and qRT-PCR analysis results verified the stronger expression of Sox9, aggrecan (ACAN), and collagen II in rabbit BMSCs treated with Shh or TGF-β in a microgravity environment. Low levels of chondrogenic hypertrophy, osteogenesis, and adipogenesis-related factors were detected in all groups. After transplantation in vivo, histological analysis revealed a significant improvement in cartilage and subchondral repair in the Shh transfection group.ConclusionsThese results suggested that Shh signalling promoted chondrogenesis in rabbit BMSCs under microgravity conditions equivalent to TGF-β, and improved the early stages of the repair of cartilage and subchondral defects. Furthermore, RCCS provided a dynamic culture microenvironment conducive for cell proliferation, aggregation and differentiation.

A Collagen and Silk Scaffold for Improved Healing of the Tendon and Bone Interface in a Rabbit Model.

BackgroundThe study aimed to develop a novel orthopedic surgical scaffold made of collagen and silk to repair the tendon and bone interface, and to investigate its influence on tendon and bone healing in a rabbit model.Material/MethodsFour types of surgical scaffold were prepared, including a random collagen scaffold (RCS), an aligned collagen scaffold (ACS), a random collagen scaffold combined with knitted silk (RCSS), and an aligned collagen scaffold combined with knitted silk (ACSS). Rabbit bone marrow stem cells (BMSCs) were cultured and seeded onto the RCS and ACS scaffold. The animal model included four-month-old female New Zealand White rabbits (N=20) that underwent drilling into the rotator cuff of the left supraspinatus muscle tendon, randomized into the ACSS and RCSS groups.ResultsRabbit BMSCs adhered to and proliferated on the RCS and ACS in vitro. Transcription levels of the COL I, COL III, and tenascin (TCN) genes were significantly increased in the ACS group compared with the RCS group. Transcription levels of COL I, runt-related transcription factor-2 (RUNX-2) and bone morphogenetic protein-2 (BMP-2) were significantly increased in the RCS group compared with the ACS group. RCSS and ACSS implanted in the rabbit models for eight weeks resulted in more regenerative tissue in the RCSS group compared with the ACSS group, with new cartilage at the tendon and bone interface at 12 weeks.ConclusionsA collagen and silk scaffold improved healing of the tendon and bone interface in a rabbit model.

Evaluation of silymarin/duck's feet-derived collagen/hydroxyapatite sponges for bone tissue regeneration

Tissue engineered scaffolds, made of natural derived materials, have the potential to be used in bone regeneration fields due to the biocompatible and biodegradable features. In this study, we propose duck's feet-derived collagen (DC) sponges blended with hydroxyapatite (HAp), incorporated with different concentrations of silymarin (Smn), for improved bone regeneration. The morphological and structural properties of DC/HAp and DC/HAp loaded with 25, 50 and 100 μM of Smn sponges were analyzed using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). In vitro evaluations were carried out on rabbit bone marrow stem cells (rBMSCs) using MTT assay for cell proliferation, ALP assay for osteogenic differentiation and reverse transcription-polymerase chain reaction (RT-PCR) for expression of mRNAs. For the evaluation of new bone formation in vivo, histological analysis and micro computed tomography (μCT) were used. Preliminary results, on Smn/DC/HAp morphology and mechanical properties, showed an interconnected porosity suitable for cells ingrowth and a higher compressive strength with the presence of Smn. Similarly, the cells proliferation and ALP activity modulation were positively influenced by the Smn content. Especially, the 100 μM Smn/DC/HAp sponge efficiently enhances the rBMSCs adhesion, growth and gene expression of osteogenic markers. The enhanced osteoinductive effects of sponges blended with Smn were confirmed using μ-CT and histological evaluations. In conclusion, results suggest that collagen sponges represent an excellent environment for cells growth and proliferation, while Smn plays an important role to improve materials osteogenic properties.

In Situ Articular Cartilage Regeneration through Endogenous Reparative Cell Homing Using a Functional Bone Marrow-Specific Scaffolding System.

In situ tissue regeneration by homing endogenous reparative cells to the injury site has been extensively researched as a promising alternative strategy to facilitate tissue repair. In this study, a promising scaffolding system DCM-RAD/SKP, which integrated a decellularized cartilage matrix (DCM)-derived scaffold with a functionalized self-assembly Ac-(RADA)4-CONH2/Ac-(RADA)4GGSKPPGTSS-CONH2 (RAD/SKP) peptide nanofiber hydrogel, was designed for repairing rabbit osteochondral defect. In vitro experiments showed that rabbit bone marrow stem cells migrated into and have higher affinity toward the functional scaffolding system DCM-RAD/SKP than the control scaffolds. One week after in vivo implantation, the functional scaffolding system DCM-RAD/SKP facilitated the recruitment of endogenous mesenchymal stem cells within the defect site. Moreover, gene expression analysis indicated that the DCM-RAD/SKP promoted chondrogenesis of the recruited cells. In vivo results showed that the DCM-RAD/SKP achieved superior hyaline-like cartilage repair and successful subchondral bone reconstruction. By contrast, the control groups mostly led to fibrous tissue repair. These findings indicate that the DCM-RAD/SKP can recruit endogenous stem cells into the site of cartilage injury and promote differentiation of the infiltrating cells into the chondrogenic lineage, holding great potential as a one-step surgery strategy for cartilage repair.

Influence of double gene recombinant vector transfected alcohol-induced rabbit stem cells on expressions of adipogenic and osteogenic genes

Objective To investigate the influence of transfection with double gene recombinant vector on the expression of adipogenic and osteogenic genes in alcohol-induced rabbit stem cells. Methods The rabbit bone marrow stem cells (BMSCs) of third generation were randomly divided into 6 groups: (1) normal group (BMSCs given no special treatment); (2) model group (MBSCs given 0.09 mol/L ethanol, no gene transfection); (3) unrelated sequence group (BMSCs givne 10 μl unrelated sequence transfection); (4) siPPARγ group (BMSCs given 10 μl siPPARγ gene vector transfection); (5) exCGRP group [BMSCs given 10 μl calcitonin gene-related peptide (CGRP) gene transfection]; (6) double gene group (BMSCs given 10 μl double gene recombinant vector transfection). Except the normal group, the final mass concentration of 0.09 mol/L ethanol was added to the remaining groups of the cells after transfection and every time the liquid was changed. The expression of PPARγ, CGRP, runt related transcription factor-2 (Runx2) and Osteocalcin mRNA and their proteins was detected at 7th and 14th day of the experiment. Results At 7th day, the expression levels of PPARγ mRNA and its protein in groups of double gene, siPPARγ and normal were low (0.329±0.037 and 0.162±0.013, 0.413±0.045 and 0.174±0.023, 0.376±0.042 and 0.169±0.021), significantly lower than those in model group (0.672±0.079 and 0.871±0.096), unrelated sequence group (0.674±0.083 and 0.823±0.089), and exCGRP group (0.683±0.079 and 0.839±0.091), and the differences were statistically significant (P=0.000). The expression levels of CGRP mRNA and its protein in groups of double gene and exCGRP were high, significantly higher than those in model group, unrelated sequence group, siPPARγ group and normal group, and the differences were statistically significant (P=0.000). The expression levels of Runx2 mRNA, Osteocalcin mRNA and theirs proteins in groups of double gene, exCGRP, siPPARγ and normal were high, those in double gene group were significantly higher than those in model group and unrelated sequence group, and higher than those in groups of exCGRP, siPPARγ and normal, and the differences were statistically significant (P=0.000). At 14th day, the expression levels in all groups were consistent with those at the 7th day. Conclusion Double gene recombinant vector transfected alcohol-induced rabbit stem cells can effectively inhibit the expression of adipogenic gene and up-regulate the expression of osteogenic genes in rabbit stem cells, which is more significant than single gene action. Key words: Double gene recombinant vector; Transfect; Alcohol; Stem cells; Adipogenic genes; Osteogenic genes; Gene expression

A novel bioactive osteogenesis scaffold delivers ascorbic acid, β-glycerophosphate, and dexamethasone in vivo to promote bone regeneration.

Ascorbic acid, β-glycerophosphate, and dexamethasone have been used in osteogenesis differentiation medium for in vitro cell culture, nothing is known for delivering these three bioactive compounds in vivo. In this study, we synthesized a novel bioactive scaffold by combining these three compounds with a lysine diisocyanate-based polyurethane. These bioactive compounds were released from the scaffold during the degradation process. The cell culture showed that the sponge-like structure in the scaffold was critical in providing a large surface area to support cell growth and all degradation products of the polymer were non-toxic. This bioactive scaffold enhanced the bone regeneration as evidenced by increasing the expression of three bone-related genes including collagen type I, Runx-2 and osteocalcin in rabbit bone marrow stem cells (BMSCs) in vitro and in vivo. The osteogenesis differentiation of BMSCs cultured in this bioactive scaffold was similar to that in osteogenesis differentiation medium and more extensive in this bioactive scaffold compared to the scaffold without these three bioactive compounds. These results indicated that the scaffold containing three bioactive compounds was a good osteogenesis differentiation promoter to enhance the osteogenesis differentiation and new bone formation in vivo.

Scaffold design for artificial tissue with bone marrow stem cells

ObjectiveThe aim of this study was to test polymeric materials (collagen, fibrin, polyimide film, and polylactic acid) for single- and multi-layer scaffold formation. Materials and methodsIn our study, we used rabbit bone marrow stem cells (rBMSCs) and human mesenchymal stem cells (hMSCs) with materials of a different origin for the formation of an artificial scaffold, such as a collagen scaffold, fibrin scaffold produced from clotted rabbit plasma, electrospun poly(lactic acid) (PLA) mats, polyimide film (PI), and the combination of the latter two. Cell imaging was performed 3–14 days after cell cultivation in the scaffolds. Time-lapse imaging was used to determine hMSC mobility on the PI film. ResultsCell incorporation in collagen and clotted fibrin scaffolds was evaluated after 2-week cultivation in vitro. Histological analysis showed that cells penetrated only external layers of the collagen scaffold, while the fibrin clot was populated with rBMSCs through the entire scaffold thickness. As well, cell behavior on the laser micro-structured PI film was analyzed. The mobility of hMSCs on the smooth PI film and the micro-machined surface was 20±2μm/h and 18±4μm/h, respectively. After 3-day cultivation, hMSCs were capable of spreading through the whole 100±10μm-thick layer of the electrospun PLA scaffold and demonstrated that the multilayer scaffold composed of PI and PLA materials ensured a suitable environment for cell growth. ConclusionsThe obtained results suggest that electrospinning technology and femtosecond laser micro-structuring could be employed for the development of multi-layer scaffolds. Different biopolymers, such as PLA, fibrin, and collagen, could be used as appropriate environments for cell inhabitation and as an inner layer of the multi-layer scaffold. PI could be suitable as a barrier blocking cell migration from the scaffold. However, additional studies are needed to determine optimal parameters of inner and outer scaffold layers.

Gene expression modulation in TGF-β3-mediated rabbit bone marrow stem cells using electrospun scaffolds of various stiffness.

Tissue engineering has recently evolved into a promising approach for annulus fibrosus (AF) regeneration. However, selection of an ideal cell source, which can be readily differentiated into AF cells of various regions, remains challenging because of the heterogeneity of AF tissue. In this study, we set out to explore the feasibility of using transforming growth factor-β3-mediated bone marrow stem cells (tBMSCs) for AF tissue engineering. Since the differentiation of stem cells significantly relies on the stiffness of substrate, we fabricated nanofibrous scaffolds from a series of biodegradable poly(ether carbonate urethane)-urea (PECUU) materials whose elastic modulus approximated that of native AF tissue. We cultured tBMSCs on PECUU scaffolds and compared their gene expression profile to AF-derived stem cells (AFSCs), the newly identified AF tissue-specific stem cells. As predicted, the expression of collagen-I in both tBMSCs and AFSCs increased with scaffold stiffness, whereas the expression of collagen-II and aggrecan genes showed an opposite trend. Interestingly, the expression of collagen-I, collagen-II and aggrecan genes in tBMSCs on PECUU scaffolds were consistently higher than those in AFSCs regardless of scaffold stiffness. In addition, the cell traction forces (CTFs) of both tBMSCs and AFSCs gradually decreased with scaffold stiffness, which is similar to the CTF change of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that tBMSCs had strong tendency to differentiate into various types of AF cells and presented gene expression profiles similar to AFSCs, thereby establishing a rationale for the use of tBMSCs in AF tissue engineering.