Subcutaneous Implantation In Rats Research Articles

Matrix levels of metalloproteinase-2 (MMP-2) and toxicity evaluation of carbonate apatite-based endodontic sealer in rat subcutaneous implantation

ObjectiveThe aims of this study is to investigate the matrix metalloproteinase-2 (MMP-2) levels and the toxicity evaluation to determine the safety of carbonate apatite material that will be used as an endodontic sealer. MethodsCarbonate apatite as endodontic sealer material is fabricated with a size of 2 × 2 × 1 mm, sterilized, and then implanted in the subcutaneous area on the murine back. Total of 28 rats was divided into 2 groups: the implantation group and the control group (positive and negative). We observed behavior, macroscopic and microscopic images (through hematoxylin-eosin/HE staining) and MMP-2 levels in the serum (through ELISA examination) to determine the reaction of implant material in experimental animals. ResultsBehavioral test shows impaired motor function within 2–24 h in all rats, autonomic behavioral test does not show any disturbance, macroscopic images show appearance of tumor and rubor within 2–72 h in all rats, microscopic images shows an increase in the mean neutrophils number with the highest point located in the 24 h (3.92×103/μL) and lymphocytes with the highest point in the 14 days (15.31×103/μL). The average value of MMP-2 levels of the negative control group was 1.33 ng/ml, 1.29 ng/ml of the positive control group, and 7.32 ng/ml of the implantation group. Level of MMP-2 in rats with visible implants increased on day 3 (0.75 ng/ml), day 10 (1.49 ng/ml) and day 14 (17.67 ng/ml). ConclusionThe implantation of carbonate apatite material did not cause behavioral disorders or abnormalities in the tissues surrounding of the implant site and did not show signs of toxicity or death.

Injectable ECM hydrogel for delivery of BMSCs enabled full-thickness meniscus repair in an orthotopic rat model

Meniscal injuries have poor intrinsic healing capability and are associated with the development of osteoarthritis. Decellularized meniscus extracellular matrix (mECM) has been suggested to be efficacious for the repair of meniscus defect. However, main efforts to date have been focused on the concentration, crosslinking density and anatomical region dependence of the mECM hydrogels on regulation of proliferation and differentiation of adult mesenchymal stem cells (MSCs) in vitro 2D or 3D culture. A systematic investigation and understanding of the effect of mECM on encapsulated MSCs response and integrative meniscus repair by in vivo rat subcutaneous implantation and orthotopic meniscus injury model will be highly valuable to explore its potential for clinical translation. In this study, we investigated the in situ delivery of rat BMSCs in an injectable mECM hydrogel to a meniscal defect in a SD rat model. Decellularized mECM retained essential proteoglycans and collagens, and significantly upregulated expression of fibrochondrogenic markers by BMSCs versus collagen hydrogel alone in vitro 3D cell culture. When applied to an orthotopic model of meniscal injury in SD rat, mECM is superior than collagen I scaffold in reduction of osteophyte formation and prevention of joint space narrowing and osteoarthritis development as evidenced by histology and micro-CT analysis. Taken together, these results indicate mECM hydrogel is a highly promising carrier to deliver MSCs for long-term repair of meniscus tissue, while preventing the development of osteoarthritis.

Effect of Ethylene Oxide Sterilization on Polyvinyl Alcohol Hydrogel Compared with Gamma Radiation.

This study investigated the effects of terminal sterilization of polyvinyl alcohol (PVA) biomaterials using clinically translatable techniques, specifically ethylene oxide (EtO) and gamma (γ) irradiation. While a few studies have reported the possibility of sterilizing PVA with γ-radiation, the use of EtO sterilization of PVA requires additional study. PVA solutions were chemically crosslinked with trisodium trimetaphosphate and sodium hydroxide. The three experimental groups included untreated control, EtO, and γ-irradiation, which were tested for the degree of swelling and water content, and mechanical properties such as radial compliance, longitudinal tensile, minimum bend radius, burst pressure, and suture retention strength. In addition, samples were characterized with scanning electron microscopy, differential scanning calorimetry, X-ray photoelectron spectroscopy, and water contact angle measurements. Cell attachment was assessed using the endothelial cell line EA.hy926, and the sterilized PVA cytotoxicity was studied with a live/dead stain. Platelet and fibrin accumulation was measured using an ex vivo shunt baboon model. Finally, the immune responses of PVA implants were analyzed after a 21-day subcutaneous implantation in rats and a 30-day implantation in baboon. EtO sterilization reduced the PVA graft wall thickness, its degree of swelling, and water content compared with both γ-irradiated and untreated PVA. Moreover, EtO sterilization significantly reduced the radial compliance and increased Young's modulus. EtO did not change PVA hydrophilicity, while γ-irradiation increased the water contact angle of the PVA. Consequently, endothelial cell attachment on the EtO-sterilized PVA showed similar results to the untreated PVA, while cell attachment significantly improved on the γ-irradiated PVA. When exposing the PVA grafts to circulating whole blood, fibrin accumulation of EtO-sterilized PVA was found to be significantly lower than γ-irradiated PVA. The immune responses of γ-irradiated PVA, EtO-treated PVA, and untreated PVA were compared. Implanted EtO-treated PVA showed the least MAC387 reaction. The terminal sterilization methods in this study changed PVA hydrogel properties; nevertheless, based on the characterizations performed, both sterilization methods were suitable for sterilizing PVA. We concluded that EtO can be used as an alternative method to sterilize PVA hydrogel material. Impact statement Polyvinyl alcohol (PVA) hydrogels have been used for a variety of tissue replacements, including neural, cardiac, meniscal, cartilage, muscle, pancreatic, and ocular applications. In addition, PVA can be made into a tubular shape and used as a small-diameter vascular graft. Ethylene oxide (EtO) is one of the Food and Drug Administration-approved methods for sterilization, but its effect on PVA has not been studied extensively. The outcome of this study provides the effects of EtO and γ-irradiation of PVA grafts on both the material properties and the in vivo responses, particularly for vascular applications. Knowledge of these effects may ultimately improve the success rate of PVA vascular grafts.

InVivo Disintegration and Bioresorption of a Nacre-Inspired Graphene-Silk Film Caused by the Foreign-Body Reaction.

SummaryGraphene-based substrates are emerging as a promising functional platform for biomedical applications. Although dispersible graphene sheets have been demonstrated to be biodegradable, their assembled macroscopic architectures are biopersistent because of strong π-π interactions. In this study, we developed a nacre-inspired graphene-silk nanocomposite film by vacuum filtration with a subsequent green chemical reduction procedure. The “brick-and-mortar” architecture not only ensures the mechanical and electrical properties of the film but also endows it with disintegrable and bioresorbable properties following rat subcutaneous implantation. Furthermore, covalent cross-linking leads to the formation of graphene with decreased interlayer spacing, which effectively prolongs the residence time in vivo. We found that enzymatic treatment created microcracks on the film surface and that the foreign-body reaction was involved in the deformation, delamination, disintegration, and phagocytosis processes of the nanocomposite films. This bioinspired strategy paves the way for the development of high-performance graphene-based macroscopic biomaterials with tunable bioresorbability.

Study on the osteogenesis of rat mesenchymal stem cells and the long-term antibacterial activity of Staphylococcus epidermidis on the surface of silver-rich TiN/Ag modified titanium alloy.

The main causes of failure of orthopedic implants are infection and poor bone ingrowth. Surface modification of the implants to allow for long-term antibacterial and osteogenic functions is an effective solution to prevent failure of the implants. We developed silver-rich TiN/Ag nano-multilayers on the surface of titanium alloy with different doses of Ag+ . The antibacterial stability and osteogenesis of the silver-rich surface were determined by evaluating the adhesion and proliferation of Staphylococcus epidermidis, and the adhesion, proliferation, alkaline phosphatase activity, extracellular matrix mineralization, and the expression level of genes involved in osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs). The results demonstrated that the antibacterial rates (Ra) of 5 × 1016 -Ag, 1 × 1017 -Ag, 5 × 1017 -Ag, and 1 × 1018 -Ag were respectively 46.21%, 85.66%, 94.99%, 98.48%, and 99.99%. After subcutaneous implantation in rats or immersion in phosphate buffered saline for up to 12 weeks, the silver-rich surface of the titanium alloy showed long-term stable inhibition of Staphylococcus epidermidis. Furthermore, in vitro and in vivo studies indicated that the Ag-implanted titanium did not show apparent cytotoxicity and that lower Ag+ implanted groups (5 × 1016 -Ag, 1 × 1017 -Ag) had better viability and biological safety when compared with higher Ag+ implanted groups. In addition, when compared with the Ti6Al4V-group, all Ag-implanted groups exhibited enhanced osteogenic indicators in rat BMSCs. Regarding osteogenic indicators, the surfaces of the 5 × 1017 -Ag group had better osteogenic effects than those of other groups. Therefore, the proper dose of Ag+ implanted TiN/Ag nano-multilayers may be one of the options for the hard tissue replacement materials with antibacterial activity and osteogenic functions.

Curcumin-crosslinked acellular bovine pericardium for the application of calcification inhibition heart valves

Glutaraldehyde (GA) crosslinked bovine or porcine pericardium tissues exhibit high cell toxicity and calcification in the construction of bioprosthetic valves, which accelerate the failure of valve leaflets and motivate the exploration for alternatives. Polyphenols, including curcumin, procyanidin and quercetin, etc, have showed great calcification inhibition potential in crosslinking collagen and elastin scaffolds. Herein, we developed an innovative phenolic fixing technique by using curcumin as the crosslinking reagent for valvular materials. X-ray photoelectron spectroscopy and Fourier transform infrared spectrometry assessments confirmed the hydrogen bond between curcumin and acellular bovine pericardium. Importantly, the calcification inhibition capability of the curcumin-crosslinked bovine pericardium was proved by the dramatically reduced Ca2+ content in the curcumin-fixed group in in vitro assay, a juvenile rat subcutaneous implants model, as well as an osteogenic differentiation model. In addition, the results showed that the curcumin-fixed bovine pericardium exhibited better performance in the areas of mechanical performance, hemocompatibility and cytocompatibility, in comparison with the GA group and the commercialized product. In summary, we demonstrated that curcumin was a feasible crosslinking reagent to fix acellular bovine pericardium, which showed great potential for biomedical applications, particularly in cardiovascular biomaterials with calcification inhibition capacity.

Additive manufacturing of biodegradable porous orthopaedic screw

Advent of additive manufacturing in biomedical field has nurtured fabrication of complex, customizable and reproducible orthopaedic implants. Layer-by-layer deposition of biodegradable polymer employed in development of porous orthopaedic screws promises gradual dissolution and complete metabolic resorption thereby overcoming the limitations of conventional metallic screws. In the present study, screws with different pore sizes (916 × 918 μm to 254 × 146 μm) were 3D printed at 200 μm layer height by varying printing parameters such as print speed, fill density and travel speed to augment the bone ingrowth. Micro-CT analysis and scanning electron micrographs of screws with 45% fill density confirmed porous interconnections (40.1%) and optimal pore size (259 × 207 × 200 μm) without compromising the mechanical strength (24.58 ± 1.36 MPa). Due to the open pore structure, the 3D printed screws showed increased weight gain due to the deposition of calcium when incubated in simulated body fluid. Osteoblast-like cells attached on screw and infiltrated into the pores over 14 days of in vitro culture. Further, the screws also supported greater human mesenchymal stem cell adhesion, proliferation and mineralized matrix synthesis over a period of 21 days in vitro culture as compared to non-porous screws. These porous screws showed significantly increased vascularization in a rat subcutaneous implantation as compared to control screws. Porous screws produced by additive manufacturing may promote better osteointegration due to enhanced mineralization and vascularization.

Optimizing Anisotropic Polyurethane Scaffolds to Mechanically Match with Native Myocardium.

Biodegradable cardiac patch is desirable to possess mechanical properties mimicking native myocardium for heart infarction treatment. We fabricated a series of anisotropic and biodegradable polyurethane porous scaffolds via thermally induced phase separation (TIPS) and tailored their mechanical properties by using various polyurethanes with different soft segments and varying polymer concentrations. The uniaxial mechanical properties, suture retention strength, ball-burst strength, and biaxial mechanical properties of the anisotropic porous scaffolds were optimized to mechanically match native myocardium. The optimal anisotropic scaffold had a ball burst strength (20.7 ± 1.5 N) comparable to that of native porcine myocardium (20.4 ± 6.0 N) and showed anisotropic behavior close to biaxial stretching behavior of the native porcine myocardium. Furthermore, the optimized porous scaffold was combined with a porcine myocardium-derived hydrogel to form a biohybrid scaffold. The biohybrid scaffold showed morphologies similar to the decellularized porcine myocardial matrix. This combination did not affect the mechanical properties of the synthetic scaffold alone. After in vivo rat subcutaneous implantation, the biohybrid scaffolds showed minimal immune response and exhibited higher cell penetration than the polyurethane scaffold alone. This biohybrid scaffold with biomimetic mechanics and good tissue compatibility would have great potential to be applied as a biodegradable acellular cardiac patch for myocardial infarction treatment.

Epoxy Chitosan-Crosslinked Acellular Bovine Pericardium with Improved Anti-calcification and Biological Properties.

Glutaraldehyde (GA) was conventionally used to crosslink bovine pericardium to prepare bioprosthetic heart valves (BHVs), which usually fail within 10 years because of valve deterioration and calcification. To overcome the high cytotoxicity and severe calcification of GA-crosslinked BHVs, a quaternary ammonium salt of epoxy chitosan (epoxy group-modified 3-chlorine-2-hydroxypropyl trimethyl chitosan, abbreviated as "eHTCC") was developed to modify the acellular bovine pericardium to substitute GA and improve its anti-calcification and biocompatible properties. Mechanical test, enzymatic stability test, blood compatibility assay, and cytocompatibility assay were used to investigate its mechanical property and biocompatibility. The anti-calcification effect of the eHTCC-modified bovine pericardium (eHTCC-BP) was assessed by in vitro assay and rat subcutaneous implantation assay. The results showed that eHTCC-BP could improve the mechanical properties and anti-enzymolysis ability of BP, as well as retain the original three-dimensional structure, compared with the uncrosslinked-BP group. Moreover, the in vivo calcification level of the eHTCC-BP group was much lower than that of the GA-BP group, which was 5.1% (2 weeks), 2.3% (4 weeks), and 0.8% (8 weeks) of the GA-BP group. In summary, this study demonstrated that eHTCC could be a potential crosslinking agent for the extracellular matrix for its favorable crosslinking effects, anti-enzymolysis, anti-calcification, and biocompatibility.

In vivo safety assessment of a bio-inspired bone adhesive

A new class of materials, bone adhesives, could revolutionise the treatment of highly fragmented fractures. We present the first biological safety investigation of a bio-inspired bone adhesive. The formulation was based upon a modified calcium phosphate cement that included the amino acid phosphoserine. This material has recently been described as substantially stronger than other bioresorbable calcium phosphate cements. Four adhesive groups with the active substance (phosphoserine) and two control groups without phosphoserine were selected for in vitro and in vivo biocompatibility testing. The test groups were subject for cell viability assay and subcutaneous implantation in rats that was followed by gene expression analysis and histology assessment after 6 and 12 weeks. All adhesive groups supported the same rate of cell proliferation compared to the α-TCP control and had viability between 45–64% when compared to cell control. There was no evidence of an increased immune response or ectopic bone formation in vivo. To conclude, this bio-inspired bone adhesive has been proven to be safe, in the present study, without any harmful effects on the surrounding soft tissue.

Osteoinductive potential of highly porous polylactide granules and Bio-Oss impregnated with low doses of BMP-2

Existing osteoplastic materials based on osteoconductive matrices lose their relevance. In this connection, to find the most effective and biocompatible carriers for osteoinductors delivery is an important task. Among the existing growth factors BMP-2 is the most effective and researched. Highly porous polylactide (PLA) granules and granular deproteinized bone material Bio-Oss were studied as carriers. Highly porous PLA granules were more effective than Bio-Oss. When using the minimum effective dose of BMP-2 for rats of 10 μg/ml impregnated in highly porous PLA granules, osteogenesis was induced in the calvarial critical-size defect and 18 ± 8% of the defect was filled with a new bone. There were no inflammatory reactions in the PLA granules implantation area. During subcutaneous implantation in rats it was shown that giant multinuclear cells took part in the resorption of the material. Their number was statistically significantly higher in the PLA granule implantation area than Bio-Oss and amounted to 71 ± 23 versus 30 ± 8 cells per 1 mm2. The obtained data showed that highly porous PLA granules were a promising basis for osteoinductive osteoplastic materials.

Biomimetic gradient scaffold of collagen–hydroxyapatite for osteochondral regeneration

Osteochondral defects remain a major clinical challenge mainly due to the combined damage to the articular cartilage and the underlying bone, and the interface between the two tissues having very different properties. Current treatment modalities have several limitations and drawbacks, with limited capacity of restoration; however, tissue engineering shows promise in improving the clinical outcomes of osteochondral defects. In this study, a novel gradient scaffold has been fabricated, implementing a gradient structure in the design to mimic the anatomical, biological and physicochemical properties of bone and cartilage as closely as possible. Compared with the commonly studied multi-layer scaffolds, the gradient scaffold has the potential to induce a smooth transition between cartilage and bone and avoid any instability at the interface, mimicking the natural structure of the osteochondral tissue. The scaffold comprises a collagen matrix with a gradient distribution of low-crystalline hydroxyapatite particles. Physicochemical analyses confirmed phase and chemical compositions of the gradient scaffold and the distribution of the mineral phase along the gradient scaffold. Mechanical tests confirmed the gradient of stiffness throughout the scaffold, according to its mineral content. The gradient scaffold exhibited good biological performances both in vitro and in vivo. Biological evaluation of the scaffold, in combination with human bone-marrow–derived mesenchymal stem cells, demonstrated that the gradient of composition and stiffness preferentially increased cell proliferation in different sub-regions of the scaffold, according to their high chondrogenic or osteogenic characteristics. The in vivo biocompatibility of the gradient scaffold was confirmed by its subcutaneous implantation in rats. The gradient scaffold was significantly colonised by host cells and minimal foreign body reaction was observed. The scaffold’s favourable chemical, physical and biological properties demonstrated that it has good potential as an engineered osteochondral analogue for the regeneration of damaged tissue.

The toughness chitosan-PVA double network hydrogel based on alkali solution system and hydrogen bonding for tissue engineering applications

Biocompatibility and mechanical properties have always been important indicators for the application of hydrogel materials in tissue engineering. In this work, a high strength and toughness chitosan-poly (vinyl alcohol) (PVA) DN (double network) hydrogel based on multiple hydrogen bonding interactions was prepared by applying the simple freezing-heating alternate treatment to the chitosan-PVA alkaline solution. The PVA first network was prepared by freeze crystallization, and the chitosan second network was constructed by raising the chitosan/KOH/urea temperature to 45 °C. The dynamic hydrogen bonding presented in the first PVA network and the second chitosan network given the hydrogel superior compressive (60%-230 KPa), tensile (152 KPa-360%), recoverability (90.77% after 5 cycles) and anti-swelling properties. The results of in vitro cell compatibility and in vivo subcutaneous implantation in rats both indicated that the chitosan-PVA DN hydrogel had the ability to promote cell attachment and wound healing. This DN hydrogel based on hydrogen bonding is expected to be applied in the tissue engineering repair. In addition, the hydrogel preparation process is simple and non-toxic, which provides a reference for the production of green and safe tissue engineering hydrogels.

Swim Bladder as a Novel Biomaterial for Cardiovascular Materials with Anti-Calcification Properties.

Calcification is a major cause of cardiovascular materials failure and deterioration, which leads to the restriction of their wide application. To develop new materials with anti-calcification capability is an urgent clinical requirement. Herein, a natural material derived from swim bladders as one promising candidate is introduced, which is prepared by decellularization and glutaraldehyde (GA) crosslinking. Data show that the swim bladder is mainly composed of collagen I, glycosaminoglycan (GAG), and elastin, especially rich in elastin, in accordance with higher elastic modulus in comparison to bovine pericardium. Moreover, the calcification of this material is proved dramatically lower than that of bovine pericardium by in vitro calcification assessments and in vivo assay using a rat subcutaneous implantation model. Meanwhile, good cytocompatibility, hemocompatibility, and enzymatic stability are demonstrated by in vitro assays. Further, a small diameter vascular graft using this material is successfully developed by rolling method and in situ implantation assay using a rat abdominal artery replacement model shows great performances in the aspect of higher patency and lower calcification. Taken together, these superior properties of swim bladder-derived material in anti-calcification, proper mechanical strength and stability, and excellent hemocompatibility and cytocompatibility endow it a great candidate as cardiovascular biomaterials.

Silk fibroin/sodium alginate composite porous materials with controllable degradation

In this study, silk fibroin (SF)/sodium alginate (SA) porous materials (PMs) with different blend ratios were generated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as crosslinking agent by a simple freeze-dried method. Degradation experiment of SF/SA PMs have been systematically investigated up to 18 days in Collagenase IA solution at 37 °C, Phosphate buffer saline (PBS) solution without enzyme was used as a control. The results showed SF/SA 50/50 PMs exhibited a lowest rate of weight loss, about 68% of the weight retained within 18 d in Collagenase IA solution. SEM images indicated Collagenase IA can degrade fibroin leading to collapse of the pure SF PMs, while SF/SA 50/50 PMs still possessed integrity of pore structure during enzyme degradation with increasing exposure time. The crystalline structure of the SF in the SF/SA PMs changed to silk II after degradation for 18 d. Furthermore, the results of the in vivo degradation by subcutaneous implantation in rats showed that all PMs can be degraded at different levels, and exhibited good subcutaneous histocompatibility to the host animals. The degradability was strongly correlated to the blend ratios in a series of SF/SA composite PMs, and insights gained in this study can serve as a guide to match desired degradation behavior with specific applications for the SF/SA composite PMs.

Autologous blood coagulum is a physiological carrier for BMP6 to induce new bone formation and promote posterolateral lumbar spine fusion in rabbits.

In the present study, we describe autologous blood coagulum (ABC) as a physiological carrier for BMP6 to induce new bone formation. Recombinant human BMP6 (rhBMP6), dispersed within ABC and formed as an autologous bone graft substitute (ABGS), was evaluated either with or without allograft bone particles (ALLO) in rat subcutaneous implants and in a posterolateral lumbar fusion (PLF) model in rabbits. ABGS induced endochondral bone differentiation in rat subcutaneous implants. Coating ALLO by ABC significantly decreased the formation of multinucleated foreign body giant cells (FBGCs) in implants, as compared with ALLO alone. However, addition of rhBMP6 to ABC/ALLO induced a robust endochondral bone formation with little or no FBGCs in the implant. In rabbit PLF model, ABGS induced new bone formation uniformly within the implant resulting in a complete fusion when placed between two lumbar transverse processes in the posterolateral gutter with an optimum dose of 100‐μg rhBMP6 per ml of ABC. ABGS containing ALLO also resulted in a fusion where the ALLO was replaced by the newly formed bone via creeping substitution. Our findings demonstrate for the first time that rhBMP6, with ABC as a carrier, induced a robust bone formation with a complete spinal fusion in a rabbit PLF model. RhBMP6 was effective at low doses with ABC serving as a physiological substratum providing a permissive environment by protecting against foreign body reaction elicited by ALLO.

Gelatin-polytrimethylene carbonate blend based electrospun tubular construct as a potential vascular biomaterial.

The present work details the fabrication of electrospun tubular scaffolds based on the biocompatible and unexploited blend of gelatin and polytrimethylene carbonate (PTMC) as a media (middle layer of blood vessel) equivalent for blood vessel regeneration. An attempt to resemble the media stimulated the selection of gelatin as a matrix (substitution for collagen) with the inclusion of the biodegradable elastomer PTMC (substitution for elastin). -The work highlights the variation of electrospinning parameters and its assiduous selection based on fiber diameter distribution and pore size distribution to obtain smooth microfibers and micropores which is reported for the first time for this blend. Electrospun conduits of gelatin-PTMC blend had fibers sized 6-8 μm and pores sized ~100-150 μm. Young's modulus of 0.40 ± 0.045 MPa was observed, resembling the tunica media of the native artery (~0.5 MPa). An evaluation of the surface properties, topography, and mechanical properties validated its physical requirements for inclusion in a vascular graft. Preliminary biological tests confirmed its minimal in-vitro toxicity and in-vivo biocompatibility. MTT assay (indirect) elucidated cell viability above 70% with scaffold extract, considered to be non-toxic according to the EN ISO-10993-5/12 protocol. The in-vivo subcutaneous implantation in rat showed a marked reduction in macrophages within 15 days revealing its biocompatibility and its possibility for host integration. This comprehensive study presents for the first time the potential of microporous electrospun gelatin and PTMC blend based tubular construct as a potential biomaterial for vascular tissue engineering. The proposed media equivalent included in a bilayer or trilayer polymeric construct can be a promising off-shelf vascular graft.

Aggregation of Nanosized Hydroxyapatite Particles and Inhibition of Cell Adhesion on this Bio-Active Material as Key Factors that Limit its Biointegration

Abstract—Different synthetic calcium phosphate compounds are used as osteoplastic materials for filling of bone defects during surgical reconstruction of bone. Synthetic nanosized hydroxyapatite is of particular interest because of its similarity to the chemical composition of bone and its osteoinductive potential. However, it is also known that nanosized hydroxyapatite can inhibit the processes of biointegration of biomaterials, although we have no clear understanding of the mechanisms that are responsible for this effect. In our in vitro studies suppression of cell adhesion on nanosized hydroxyapatite that may lead to anoikis was shown. Studies in which rat subcutaneous heterotopic implantation methods were used showed that nanosized hydroxyapatite in vivo formed a slightly resorbable agglomerate that blocks cell migration into the material, as well as neocollagenesis processes in it, including the processes of encapsulation of the material. In this case, the processes of neocollagenesis along the periphery of the sample were observed only over a dense layer of dead cells (cellular debris) surrounding the agglomerated nanosized hydroxyapatite. Addition of demineralized bone collagen matrix to nanosized hydroxyapatite as a conductive component contributed to colonization of the material by the cells, resorption of nanosized hydroxyapatite, and active construction of the neocollagen matrix. These results show that modification of nanosized hydroxyapatite with a conductive component is required in order to prevent its agglomeration and thus increase its biointegration and osteoinductive potential in the body.

P4. Autologous blood coagulum as a native carrier for rhBMP6 induces new bone formation and posterolateral lumbar spine fusion in rabbits and sheep

BACKGROUND CONTEXT RhBMP2 soaked in bovine tendon collagen and synthetic ceramics composite as well as rhBMP7/OP1 lyophilized in bovine bone collagen particles with additive carboxyl-methyl cellulose have been evaluated for posterolateral lumbar fusion (PLF). We investigated rhBMP6 delivered in autologous blood coagulum (ABC), a native carrier (scaffold), with and without allograft bone particles (ALLO) to induce new bone formation and promote posterolateral lumbar spine fusion (PLF) in rabbit and sheep. PURPOSE To evaluate the efficacy of RhBMP6 with autologous blood coagulum (ABC) alone or with ABC and allograft (ALLO) will induce a successful posterolateral spinal fusion in animal models. STUDY DESIGN/SETTING Preclinical evaluation of spine fusion in animal models. PATIENT SAMPLE Multiple experimental groups of rats, rabbits and sheep. OUTCOME MEASURES Radiographic and histologic evaluation for bone formation and spinal fusion. METHODS An autologous bone graft substitute (ABGS) was fabricated by dispersing rhBMP6 in ABC with and without ALLO and evaluated for bone induction in rat subcutaneous implants and to promote bone formation in posterolateral lumbar fusion in preclinical models. The cellular response of bone induction and the quantity and quality of newly formed bone were examined by histology, radiographs, micro-CT analyses at various time intervals. RESULTS ABGS with and without ALLO induced new bone formation in rat implants. ALLO alone induced formation of multinucleated foreign body giant cells (FBGCs). However, ALLO plus ABC decreased the formation of FBGCs. The addition of rhBMP6 to ABC/ALLO, resulted in fewer or no FBGCs. In Rabbits, the groups with ABC alone or with ALLO did not fuse. The 50 micrograms rhBMP6 group only 2 out of 4 animals successfully fused. In the 100 micrograms, 200 micrograms and 400 micrograms rhBMP6 groups all animals successfully fused. In sheep, the groups with ABC or ABC with ALLO failed to fuse. In the rhBMP6 187.5 micro grams/ml group with ABC and instrumentation fusion was achieved in 83% (10/12 animals). In the 187.5 micro grams/ml group with ABC, ALLO and instrumentation, fusion was achieved in 93% (11/12 animals). Bone formation occurred uniformly within ABGS implants where the ALLO was replaced by the newly formed bone via creeping substitution. CONCLUSIONS RhBMP6 with autologous blood coagulum (ABC) alone or with ABC and allograft (ALLO) induced a robust bone formation in posterolateral spinal fusion in preclinical models. FDA DEVICE/DRUG STATUS RhBMP6 (Not approved for this indication).

A high-strength biodegradable thermoset polymer for internal fixation bone screws: Preparation, in vitro and in vivo evaluation

Thermoset polymers synthesized from the polycondensation of glycerol with biocompatible diacids represent a promising class of absorbable materials for biomedical applications. However, the utility of these polymers for bone fixation devices is hampered due to the lack of mechanical strength. Herein we synthesized a high-strength thermoset polymer, poly(glycerol-succinate) (PGS), via a catalyst-free and solvent-free reaction. The bending strength of PGS reaches 122.01 ± 8.82 MPa, signifying its great potential for fixation devices. The degradation property of the polymer can be tuned by adjusting the monomer ratio and reaction time. Bone screws based on the PGS polymer were successfully manufactured using a lathe. In vitro evaluation showed the PGS polymer was able to well support cell adhesion and proliferation. In vivo evaluation using a rat subcutaneous implantation model showed that the inflammatory response to the polymer was mild. After the PGS screws were implanted in the rabbit femoral condyle for 12 weeks, micro-computed tomography (micro-CT) and histological analysis revealed that the screws achieved good osseointegration. Consequently, the polymer developed in current study can serve as internal fixation devices due to the proper mechanical strength, excellent biocompatibility, and feasibility of manufacturing screws.