Specifics of Porous Polymer and Xenogeneic Matrices and of Bone Tissue Regeneration Related to Their Implantation into an Experimental Rabbit Defect.

This paper provides a study of two bone substitutes: a hybrid porous polymer and an osteoplastic matrix based on a bovine-derived xenograft. Both materials are porous, but their pore characteristics are different. The osteoplastic matrix has pores of 300-600 µm and the hybrid polymer has smaller pores, generally of 6-20 µm, but with some pores up to 100 µm across. SEM data confirmed the porometry results and demonstrated the different structures of the materials. Therefore, both materials were characterized by an interconnected porous structure and provided conditions for the adhesion and vital activity of human ASCs in vitro. In an experimental model of rabbit shin bone defect, it was shown that, during the 6-month observation period, neither of the materials caused negative reactions in the experimental animals. By the end of the observation period, restoration of the defects in animals in both groups was completed, and elements of both materials were preserved in the defect areas. Data from morphological examinations and CT data demonstrated that the rate of rabbit bone tissue regeneration with the hybrid polymer was comparable to that with the osteoplastic matrix. Therefore, the hybrid polymer has good potential for use in further research and improvement in biomedical applications.

K-wire is more damaging than standard or acrylic drill bits when evaluating torsional properties of rabbit (Oryctolagus cuniculi) femurs.

OBJECTIVE: The objective of this study is to compare drilling variables and torsional mechanical properties of rabbit femora after bicortical drilling with a 1.5-mm standard surgical drill bit, acrylic drill bit, and K-wire. SAMPLES: 24 pairs of rabbit femora. METHODS: After drilling under controlled axial displacement rate, each bone was biaxially loaded in compression followed by rapid external torsion to failure. Maximum axial thrust force, maximum drill torque, integral of force and displacement, change in temperature, maximum power spectral density of the torque signal, torque vibration, and torque and angle at the yield and failure points were collected. Pre- and postyield stiffness, yield and failure energies, and postyield energy were calculated. RESULTS: The work required to drill through the cis- and transcortices (integral of force and displacement) was greater for the K-wire, followed by the acrylic and then standard drill bits, respectively. The K-wire demonstrated higher maximum torque than the drill bits at the ciscortex, and the force of drilling was significantly greater. The vibration data was greater with the acrylic and standard drill bits than the K-wire. There was no difference in torsional strength between drilling types. CLINICAL RELEVANCE: Mechanical differences exist between different drill bits and K-wire and demonstrate that the K-wire is overall more damaging than the surgical drill bit.

Seamless and early gap healing of osteochondral defects by autologous mosaicplasty combined with bioactive supramolecular nanofiber-enabled gelatin methacryloyl (BSN-GelMA) hydrogel.

Autologous mosaicplasty is a common approach used to treat osteochondral defects in clinical practice. Gap integration between host and transplanted plugs requires bone tissue reservation and hyaline cartilage regeneration without uneven surface, graft necrosis and sclerosis. However, poor gap integration is a serious concern, which eventually leads to deterioration of joint function. To deal with such complications, this study has developed a strategy to effectively enhance integration of the gap region following mosaicplasty by applying injectable bioactive supramolecular nanofiber-enabled gelatin methacryloyl (GelMA) hydrogel (BSN-GelMA). A rabbit osteochondral defect model demonstrated that BSN-GelMA achieved seamless osteochondral healing in the gap region between plugs of osteochondral defects following mosaicplasty, as early as six weeks. Moreover, the International Cartilage Repair Society score, histology score, glycosaminoglycan content, subchondral bone volume, and collagen II expression were observed to be the highest in the gap region of BSN-GelMA treated group. This improved outcome was due to bio-interactive materials, which acted as tissue fillers to bridge the gap, prevent cartilage degeneration, and promote graft survival and migration of bone marrow mesenchymal stem cells by releasing bioactive supramolecular nanofibers from the GelMA hydrogel. This study provides a powerful and applicable approach to improve gap integration after autologous mosaicplasty. It is also a promising off-the-shelf bioactive material for cell-free in situ tissue regeneration.

TGF-ß1 and -ß3 for Mesenchymal Stem Cells Chondrogenic Differentiation on Poly (Vinyl Alcohol)-Chitosan-Poly (Ethylene Glycol) Scaffold.

Transforming growth factor-beta 1 (TGF-ß1) has been reported to promote chondrogenic differentiation and proliferation in the multipotent stromal cell (MSCs), and the transforming growth factor-beta 3 (TGF-ß3) tends to be exclusively in promoting cell differentiation alone. The objective of this study was to determine the effect of TGF-ß1 and -ß3 on the MSCs chondrogenic differentiation on the poly (vinyl alcohol)-chitosan-poly (ethylene glycol) (PVA-NOCC-PEG) scaffold, compared with that of monolayer and pellet cultures. In this study, P2 rabbit bone marrow-derived MSCs were seeded either on the untreated six-well plate (for monolayer culture) or onto the PVA-NOCC-PEG scaffold or cultured as a pellet culture. The cultures were maintained in a chemically defined serum-free medium supplemented with 10 ng/mL of either TGF-ß1 or TGF-ß3. Cell viability assay, biochemical assay, and real-time polymerase chain reaction were performed to determine the net effect of cell proliferation and chondrogenic differentiation of each of the growth factors. The results showed that the PVA-NOCC-PEG scaffold enhanced MSCs cell proliferation from day 12 to 30 (p < 0.05); however, no significant differences were observed in the cell proliferation between the cultures supplemented with or without TGF-ß1 and TGF-ß3 (p > 0.05). In terms of chondrogenic differentiation, the PVA-NOCC-PEG scaffold augmented the GAGs secretion in MSCs and the mRNA expression levels of Sox9, Col2a1, Acan, and Comp were elevated (p < 0.05). However, there was no significant difference between both the TGF-ß1 and TGF-ß3-treated groups (p > 0.05). In conclusion, TGF-ß1 and TGF-ß3 enhanced the chondrogenic differentiation of MSCs seeded on the PVA-NOCC-PEG scaffold; however, there was no significant difference between the effect of TGF-ß1 and TGF-ß3. Impact statement Transforming growth factor-beta (TGF-ß) superfamily members is a key requirement for the in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs). In this study, the effects of TGF-ß1 and -ß3 on MSC chondrogenic differentiation and proliferation on a novel three-dimensional scaffold, the poly(vinyl alcohol)-chitosan-poly(ethylene glycol) (PVA-NOCC-PEG) scaffold, was evaluated. In this study, the results showed both TGF-ß1 and TGF-ß3 can enhance the chondrogenic differentiation of MSCs seeded on the PVA-NOCC-PEG scaffold.

Conformational Changes in Proteins Caused by High-Pressure Homogenization Promote Nanoparticle Formation in Natural Bone Aqueous Suspension.

As a natural calcium resource, animal bone needs to be miniaturized to the nanoscale to improve palatability and absorption capacity. To explore the mechanism of high-pressure homogenization (HPH) in preparing natural bone aqueous nanosuspensions, the relationships between the changes in protein conformation, solubility and quality characteristics of rabbit bone aqueous suspensions (RBAS) prepared by different HPH cycles were studied. The results showed that the improvements in particle size, stability and calcium solubility of RBASs could be mainly attributed to the improvement of protein solubility induced by the changes in protein conformation. HPH treatment led to the denaturation and degradation of protein in rabbit bone, generating soluble peptides and improving the stability of the suspensions by enhancing the surface charge of the particles. When collagen as the main protein was partially degraded, the hydroxyapatite in the bone was crushed into tiny particles. The increase in the particle-specific surface area led to the release of calcium ions, which chelated with the peptides to produce peptide calcium. However, excessive HPH treatment caused the production of protein macromolecular aggregates and affected the quality of RBASs. This study is helpful to promote the application of HPH technology in animal bone nanoprocessing.

Fibronectin-coating enhances attachment and proliferation of mesenchymal stem cells on a polyurethane meniscal scaffold.

INTRODUCTION: Partial meniscectomy is one of the most common surgical strategy for a meniscal injury, but sometimes, patients complain of knee pain due to an overload in the ablated compartment. In these cases, implantation of tissue engineering scaffold could be indicated. Currently, two commercial scaffolds, based on collagen or polycaprolactone-polyurethane (PCL-PU), are available for meniscus scaffolding. In short term follow-up assessments, both showed clinical improvement and tissue formation. However, long-term studies carried out in PCL-PU showed that the new tissue decreased in volume and assumed an irregular shape. Moreover, in some cases, the scaffold was totally reabsorbed, without new tissue formation.Mesenchymal stem cells (MSCs) combined with scaffolds could represents a promising approach for treating meniscal defects because of their multipotency and self-renewal. In this work, we aimed to compare the behaviour of MSCs and chondrocytes on a PCL-PU scaffold in vitro. MSCs express integrins that binds to fibronectin (FN), so we also investigate the effect of a FN coating on the bioactivity of the scaffold. METHODS: We isolated rabbit bone marrow MSCs (rBM-MSCs) from two skeletally mature New Zealand white rabbits and stablished the optimum culture condition to expand them. Then, they were seeded over non-coated and FN-coated scaffolds and cultured in chondrogenic conditions. To evaluate cell functionality, we performed an MTS assay to compare cell proliferation between both conditions. Finally, a histologic study was performed to assess extracellular matrix (ECM) production in both samples, and to compare them with the ones obtained with rabbit chondrocytes (rCHs) seeded in a non-coated scaffold. RESULTS: A culture protocol based on low FBS concentration was set as the best for rBM-MSCs expansion. The MTS assay revealed that rBM-MSCs seeded on FN-coated scaffolds have more cells on proliferation (145%; 95% CI: 107%-182%) compared with rBM-MSCs seeded on non-coated scaffolds. Finally, the histologic study demonstrated that rCHs seeded on non-coated scaffolds displayed the highest production of ECM, followed by rBM-MSCs seeded on FN-coated scaffolds. Furthermore, both cell types produced a comparable ECM pattern. CONCLUSION: These results suggest that MSCs have low capacity attachment to PCL-PU scaffolds, but the presence of integrin alpha5beta1 (FN-receptor) in MSCs allows them to interact with the FN-coated scaffolds. These results could be applied in the design of scaffolds, and might have important clinical implications in orthopaedic surgery of meniscal injuries.

Cytotoxicity assessment of bone marrow mesenchymal stem cells-cuttlebone composite scaffold / 中国组织工程研究

BACKGROUND: Repair materials for bone tissue engineering should hold good biocompatibility and degradability. There are various related studies, but the Chinese medicine composite cellular bioscaffolds are little reported. OBJECTIVE: To detect the in vitro cytotoxicity of rabbit bone marrow mesenchymal stem cells-cuttlebone bioscaffold based on the Biological Evaluation of Medical Device, and assess its cytotoxicity level in order to provide the theoretical support for its clinical application. METHODS: Bone marrow mesenchymal stem cells-cuttlebone bioscaffold extract was prepared according to an ISO standard — material area: extraction medium volume = 3-6 cm2:1 mL. L-929 cell suspension was prepared, and the cells were then cultured with a density of 1×107/L. There were three groups: positive group (DMEM medium containing phenol), experimental group (material extract), and negative group (DMEM culture medium). The absorbance value of L-929 cells was detected by MTT assay after 24, 48 and 72 hours of culture. The relative proliferation rate of cells was then calculated and the toxicity level was valued in each group. RESULTS AND CONCLUSION: The absorbance values in the experimental, negative and positive groups were not exactly same at different time points (P=0.000 < 0.01). The absorbance values in the experimental and negative groups were significantly higher than those in the positive group (P < 0.01).The cytotoxicity of bone marrow mesenchymal stem cells-cuttlebone bioscaffold was grade 1. To conclude, the bone marrow mesenchymal stem cells-cuttlebone bioscaffold has no obvious toxic effects, and meets the requirements of biomaterial application.

Effect of nano-scaled rabbit bone powder on physicochemical properties of rabbit meat batter.

BACKGROUND: To explore a new method of deep processing and to improve the value of rabbit bone, we prepared a nano-scaled rabbit bone powder by dry ball milling and compared the effect of different particle sizes of rabbit bone powder [fine-scaled (236.01 ± 5.99 µm), superfine-scaled (65.92 ± 1.71 µm), nano-scaled (502.52 ± 11.72 nm)] on the nutritional characteristics, pH, color, water-holding capacity, textural and rheological attributes of rabbit meat batter. RESULTS: The rabbit bone powder significantly affected nutritional characteristics of meat batters; in particular, the contents of calcium were increased, regardless of particle size. Additionally, the rabbit meat batter, which contained 20 g kg-1 nano-scaled rabbit bone, had the lowest centrifugal and cooking losses among the treatments. CONCLUSION: Based on the textural and rheological attributes of the rabbit meat batters, the addition of 20 g kg-1 nano-scaled rabbit bone was the best treatment. This represents an important finding with respect to the deep processing of rabbit bone in the rabbit meat industry. © 2018 Society of Chemical Industry.

Three-Dimensional Printed Scaffolds with Multipotent Mesenchymal Stromal Cells for Rabbit Mandibular Reconstruction and Engineering.

Multipotent mesenchymal stromal cells (MSC) derived from both the bone marrow and adipose tissue possess the ability to differentiate into multiple cell lineages, regulate the immune function by secreting numerous bioactive paracrine factors, and hold great potential in cell therapy and tissue engineering. When combined with three-dimensional (3D) scaffolds, MSC can be used for bone defect reconstruction and engineering. This protocol describes the isolation of bone marrow mesenchymal stromal cells (BMMSC) and adipose-tissue derived stem cells (ADSC) from rabbits for subsequent seeding on tissue-engineered 3D-printed scaffolds and transplantation into a rabbit-model with the goal of repairing large osseous mandibular defects (one quarter of the lower jaw is removed surgically). Steps to demonstrate the three cell differentiation lineage potentials of BMMSC and ADSC into osteocytes, adipocytes, and chondrocytes are described. A modified cell seeding method using syringes on scaffold is detailed. Creating a large mandibular bone defect, the rapid prototyping method to print a customized 3D-scaffold, the scaffold implantation procedure in rabbits, and microcomputed tomography (micro-CT) analysis are also described.

Collagen mimetic peptide-PEG hybrid hydrogel for 3D culture of rabbit bone marrow mesenchymal stem cells / 国际生物医学工程杂志

Objective To construct a extracellular matrix-like collagen mimetic peptide-PEG hybrid hydrogel and to study the usage of this hydrogel in 3D culture of rabbit bone marrow mesenchymal stem cells (rBMSCs).Methods The hybrid hydrogel was synthesised by conjugating the cysteine at the end of the collagen mimetic peptide with the maleimine-modified multi-arm PEG.The circular dichroism spectra were used to characterize the triple helix structure and thermal stability of the collagen mimetic peptides.The rheology test and scanning electron microscopy were used to study the gelation process,mechanical strength and internal structure of the hydrogel.The rBMSCs were embedded in the hybrid hydrogel for 3D culture.The cell compatibility of the hydrogel and its effect on differentiation of the cells was studied.Results Collagen mimetic peptides could promote spontaneous formation of triple helix structure in the natural collagen,and the thermal transition temperature was 49.4 ℃.The formation process of the collagen mimetic peptides-PEG hybrid hydrogel was rapid,in which the porous network-like fibrous structure was formed.After the encapsulation of rBMSCs within the hydrogel for 24 h,most of the cells remained viable.Gene expression analysis showed that the hybrid hydrogel could affect the differentiation of rBMSCs.Conclusions The collagen mimetic peptide-PEG hybrid hydrogel possesses the characteristics of mild preparation condition,good mechanical strength and good cell compatibility,and is favorable to chondrocyte differentiation of rBMSCs.

Osteogenic differentiation of CD271(+) cells from rabbit bone marrow cultured on three phase PCL/TZ-HA bioactive scaffolds: comparative study with mesenchymal stem cells (MSCs).

Tissue engineering is one of the major challenges of orthopedics and trauma surgery for bone regeneration. Biomaterials filled with mesenchymal stem cells (MSCs) are considered the most promising approach in bone tissue engineering. Furthermore, our previous study showed that the multi-phase poly [ε-caprolactone]/thermoplastic zein-hydroxyapatite (PCL/TZ-HA) biomaterials improved rabbit (r) MSCs adhesion and osteoblast differentiation, thus demonstrating high potential of this bioengineered scaffold for bone regeneration. In the recent past, CD271 has been applied as a specific selective marker for the enrichment of MSCs from bone marrow (BM-MSCs). In the present study, we aimed at establishing whether CD271-based enrichment could be an efficient method for the selection of rBM-MSCs, displaying higher ability in osteogenic differentiation than non-selected rBM-MSCs in an in vitro system. CD271(+) cells were isolated from rabbit bone marrow and were compared with rMSCs in their proliferation rate and osteogenic differentiation capability. Furthermore, rCD271(+) cells were tested in their ability to adhere, proliferate and differentiate into osteogenic lineage, while growing on PCL/TZ-HA scaffolds, in comparison to rMSCs. Our result demonstrate that rCD271(+) cells were able to adhere, proliferate and differentiate into osteoblasts when cultured on PCL/TZ-HA scaffolds in significantly higher levels as compared to rMSCs. Based on these findings, CD271 marker might serve as an optimal alternative MSCs selection method for the potential preclinical and clinical application of these cells in bone tissue regeneration.

Quantitative analysis of factors influencing tissue-engineered bone formation by detecting the expression levels of alkaline phosphatase and bone γ-carboxyglutamate protein 2.

Bone tissue engineering is a promising alternative approach that permits the efficient reconstruction of bone defects. There are four elements involved in bone tissue engineering technology, including the seed cells, growth factors, scaffolds and culture environment. The aim of the present study was to evaluate the effect of these factors on bone formation in tissue engineering technology by analyzing the expression of osteogenetic markers using polymerase chain reaction (PCR). Bone marrow mesenchymal stem cells (BMSCs) were extracted from the bone marrow of the bilateral tibial platform of New Zealand white rabbits. In addition, platelet-rich plasma (PRP) samples were prepared from blood extracted from the ear vein of the rabbits. A perfusion bioreactor was used to provide the culture environment, and ß-tricalcium phosphate (ß-TCP) was used to build the scaffolds. The ß-TCP scaffolds were divided into five groups and each group was treated with a different combination of the factors. Next, the composites were implanted into the rabbits. After three months, the expression levels of the new bone formation markers, alkaline phosphatase and bone γ-carboxyglutamate protein 2, were detected using quantitative reverse transcription-PCR analysis. The expression levels of the markers in the experimental groups were higher compared with the negative control group. Comparisons between the experimental groups also revealed statistical significance. Scanning electron microscopy revealed good adhesion and distribution of the BMSCs on the ß-TCP scaffold. In conclusion, the PCR results indicated that PRP, BMSCs and the bioreactor exhibited a promoting effect on bone formation.

Construction of tissue-engineered bone using a bioreactor and platelet-rich plasma.

The aim of the present study was to construct tissue-engineered bone using a bioreactor and platelet-rich plasma (PRP). Bone marrow mesenchymal stem cells (BMSCs) and ß-tricalcium phosphate (ß-TCP) were cultured in a perfusion bioreactor with PRP-containing medium for 21 days to form a BMSC-TCP composite. Rabbits were then implanted with the BMSC-TCP composite. The morphology of the implanted BMSC-TCP composite was observed three months after surgery by scanning electron microscopy and hematoxylin and eosin (H&E) staining. In addition, the expression of cluster of differentiation (CD)31 and von Willebrand factor (WF) in the implanted BMSC-TCP composite was detected using immunohistochemistry. Bone formation was determined by comprehensive testing Following culture in a perfusion bioreactor and PRP, the BMSCs adhered to the ß-TCP scaffold and the secretion of extracellular matrix was observed. The spreading and proliferation of cells was found to be enhanced on the scaffold. Furthermore, the vascular endothelial cell markers CD31 and VEF, were positively expressed. Therefore, these results suggest that tissue-engineered bone may be constructed using a bioreactor and PRP. PRP, which contains multiple growth factors, may promote vascularization of tissue-engineered bone.