The feasibility and safety of biomaterials for posterior scleral reinforcement in rabbits.

To explore the feasibility and safety of biomaterials for posterior scleral reinforcement (PSR) in rabbits. Decellularization and genipin crosslink were applied to the fresh bovine pericardium and porcine endocranium, and then mechanical properties, suture retention strength, and stability were tested. PSR operation was performed on 24 rabbit eyes using treated biological materials. Ophthalmic examination was performed regularly before and after PSR operation (1 week, 1 month, 3 months, 6 months). To evaluate the effectiveness, A ultrasound, diopter, and optical coherence tomography were conducted. General condition, fundus photograph, and pathological examination were recorded to evaluate the safety. Compared with genipin crosslinked bovine pericardium (Gen-BP) (21.29 ± 13.29 Mpa), genipin crosslinked porcine endocranium (Gen-PE) (34.85 ± 3.67 Mpa,P< 0.01) showed a closer elastic modulus to that of genipin crosslinked human sclera. There were no complications or toxic reactions directly related to the materials. Capillary hyperplasia, inflammatory cell infiltration, and collagen fiber deposition were observed, and the content of type I collagen fibers increased after PSR. Overall, the choroidal thickness of treated eyes was significantly thickened at different time points after PSR, which were 96.84 ± 21.08 µm, 96.72 ± 22.00 µm, 90.90 ± 16.57 µm, 97.28 ± 14.74 µm, respectively. The Gen-PE group showed changes that were almost consistent with the overall data. Gen-BP and Gen-PE are safe biological materials for PSR. The Gen-PE group demonstrated more significant advantages over the Gen-BP group in terms of material properties.

Advances in digital light processing of hydrogels.

Hydrogels, three-dimensional (3D) networks of hydrophilic polymers formed in water, are a significant type of soft matter used in fundamental and applied sciences. Hydrogels are of particular interest for biomedical applications, owing to their soft elasticity and good biocompatibility. However, the high water content and soft nature of hydrogels often make it difficult to process them into desirable solid forms. The development of 3D printing (3DP) technologies has provided opportunities for the manufacturing of hydrogels, by adopting a freeform fabrication method. Owing to its high printing speed and resolution, vat photopolymerization 3DP has recently attracted considerable interest for hydrogel fabrication, with digital light processing (DLP) becoming a widespread representative technique. Whilst acknowledging that other types of vat photopolymerization 3DP have also been applied for this purpose, we here only focus on DLP and its derivatives. In this review, we first comprehensively outline the most recent advances in both materials and fabrication, including the adaptation of novel hydrogel systems and advances in processing (e.g. volumetric printing and multimaterial integration). Secondly, we summarize the applications of hydrogel DLP, including regenerative medicine, functional microdevices, and soft robotics. To the best of our knowledge, this is the first time that either of these specific review focuses has been adopted in the literature. More importantly, we discuss the major challenges associated with hydrogel DLP and provide our perspectives on future trends. To summarize, this review aims to aid and inspire other researchers investigatng DLP, photocurable hydrogels, and the research fields related to them.

Collagen I gel can facilitate homogenous bone formation of adipose-derived stem cells in PLGA-beta-TCP scaffold.

Cell-based tissue engineering is thought to be a new therapy for treatment of bone defects and nonunions after trauma and tumor resection. In this study, we explore the in vitro and in vivo osteogenesis of a novel biomimetic construct fabricated by using collagen I gel to suspend rabbit adipose-derived stem cells (rASCs) into a porous poly(lactic-co-glycolic)acid-beta-tricalcium phosphate (PLGA-beta-TCP) scaffold (rASCs-COL/PLGA-beta-TCP). In vitro and in vivo studies of the rASCs-COL/PLGA-beta-TCP composite (group A) were carried out compared with the single combination of rASCs and PLGA-beta-TCP (rASCs/PLGA-beta-TCP; group B), the combination of acellular collagen I gel and PLGA-beta-TCP (COL/PLGA-beta-TCP; group C), and the PLGA-beta-TCP scaffold (group D). Composites of different groups were cultured in vitro for 2 weeks in osteogenic medium and then implanted into the autologous muscular intervals for 8 weeks. After 2 weeks of in vitro culture, alkaline phosphatase activity and extracellular matrix mineralization in group A were significantly higher than in group B (p < 0.01, n = 4). In vivo osteogenesis was evaluated by radiographic and histological analyses. The calcification level was radiographically evident in group A, whereas no apparent calcification was observed in groups B, C and D (n = 4). In group A, woven bone with a trabecular structure was formed, while in group B, only osteoid tissue was observed. Meanwhile, the bone-forming area in group A was significantly higher than in group B (p < 0.01, n = 4). No bone formation was observed in groups C or D (n = 4). In conclusion, by using collagen I gel to suspend rASCs into porous PLGA-beta-TCP scaffold, osteogenic differentiation of rASCs can be improved and homogeneous bone tissue can be successfully formed in vivo.

[Surface modification of biodegradable polymer/TCP scaffolds and related research].

Under laboratory condition, the compound materials of Poly (DL-lactic-co-glycolic acid)/Tricalcium phosphate [PLGA/TCP(L), with component ratio of 7:3] were fabricated by combining the thermally induced phase separation (TIPS) with solvent-casting particulate-leaching (SCPL) approach. On the other hand, rapid prototyping (RP) technique manufactured PLGA/TCP scaffolds [PLGA/TCP(RP)] were obtained. These two kinds of carriers were coated with collagen type I (Col I). The extracted bovine bone morphogenetic protein (bBMP) was loaded into carriers to establish biomimetic synthetic bones. PLGA/TCP(L) scaffolds, demineralized bone matrices (DBM) of bovine cancellous bone, PLGA/TCP(L) scaffolds, biomimetic synthetic bones and OsteoSet bone graft substitutes were investigated. Scanning electron microscopy revealed that the microarchitecture of PLGA/TCP(RP) scaffolds was much better than that of PLGA/TCP(L) scaffolds. The diameter of macropore of PLGA/TCP(RP) scaffold was 350 microm. The porosities of PLGA/ TCP(L) scaffolds, DBM, PLGA/TCP(RP) scaffolds and OsteoSet bone graft substitutes were 21.5%, 70.4%, 58.6% and 0%, respectively (P<0.01). Modification of PLGA/TCP scaffolds with collagen type I [PLGA/TCP(L)-Col I and PLGA/TCP(RP)-Col I] essentially increased the affinity of the carriers to bBMP. Among these synthetic materials, PLGA/TCP(RP)-Col I-bBMP composite is promising as a novel bone graft substitute due to its advanced fabrication technique, good tri-dimensional microarchitecture and ideal components.

Multilayered Scaffold with a Compact Interfacial Layer Enhances Osteochondral Defect Repair.

Repairing osteochondral defect (OCD) using advanced biomaterials that structurally, biologically, and mechanically fulfill the criteria for stratified tissue regeneration remains a significant challenge for researchers. Here, a multilayered scaffold (MLS) with hierarchical organization and heterogeneous composition is developed to mimic the stratified structure and complex components of natural osteochondral tissues. Specifically, the intermediate compact interfacial layer within the MLS is designed to resemble the osteochondral interface to realize the closely integrated layered structure. Subsequently, macroscopic observations, histological evaluation, and biomechanical and biochemical assessments are performed to evaluate the ability of the MLS of repairing OCD in a goat model. By 48 weeks postimplantation, superior hyalinelike cartilage and sound subchondral bone are observed in the MLS group. Furthermore, the biomimetic MLS significantly enhances the biomechanical and biochemical properties of the neo-osteochondral tissue. Taken together, these results confirm the potential of this optimized MLS as an advanced strategy for OCD repair.

Porous morphology, porosity, mechanical properties of poly(alpha-hydroxy acid)-tricalcium phosphate composite scaffolds fabricated by low-temperature deposition.

Tissue engineering is expected to construct complicated hominine organs composed of many different types of cells. One of the key points is the accurate controlling of scaffold material and porous morphology point by point. A new direct rapid prototyping process called low-temperature deposition manufacturing (LDM) was proposed to fabricate scaffolds. The new process integrated extrusion/jetting and phase separation and therefore could fabricate scaffolds with hierarchical porous structures creating a wonderful environment for the growth of new tissue. The interconnected computer-designed macropores allow cells in the new tissue to grow throughout the scaffold. Also, the parameter-controlled micropores let nutrition in and metabolic wastes out. The macrocellular morphology, microcellular morphology, porosity, and mechanical properties of the poly(alpha-hydroxy acid)-TCP composite scaffolds prepared by the proposed method are investigated. These scaffolds with high controllability would potentially play an important role in tissue engineering. LDM could also be combined with multinozzle deposition or cell deposition to exactly control materials or cells point by point. This might bring a breakthrough to the engineered fabrication of complicated organs.

Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans.

The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni2+-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 A resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 A, beta = 98.82 degrees.

Repair of the radial defect of rabbit with polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology.

OBJECTIVE: To evaluate the effects of repairing rabbit radial defects with polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with bovine bone morphogenetic protein (bBMP), and find new carriers for growth factors. METHODS: Polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with and without bovine BMP were used to repair the 15 mm radial defect in rabbit. Then the results of radiography, histology, scaffolds degrade rates and bone mineral density (BMD) were appraised to examine the effects at the 12th week. RESULTS: At the 12th week postoperatively, all defects treated with bBMP were radiographically repaired. No radius implanted polyester/tricalcium phosphate scaffolds without bBMP showed radiographic and histological union. At experimental groups, longitudinal alignment of lamellar structure was observed histologically at the 12th week, indicating that remodeling of regenerated bone was complete in different degree. Of the three experimental groups, the bony regeneration and remodeling of callus in poly lactide-co-glycolide/tricalcium phosphate (PLGA/TCP) group was the best. The BMD values were beyond 70% of normal value at the 12th week while the PLGA/TCP scaffolds group was the highest, and no abnormalities were observed in the surrounding soft tissue in all groups. CONCLUSIONS: Polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with bovine BMP can repair a 15 mm radial defect of rabbit. As for the results, the PLGA/TCP scaffold is ideal and better than poly L-lactide-co-D, L-lactide (PDLLA/TCP) scaffold, but the ploy L-lactic acid (PLLA/TCP) is not so good for its low degradation rates.

Fabrication of viable tissue-engineered constructs with 3D cell-assembly technique.

We have recently developed an organ manufacturing technique that enables us to form cell/biomaterial complex three-dimensional (3D) architectures in designed patterns. This technique employs a highly accurate 3D micropositioning system with a pressue-controlled syringe to deposit cell/biomaterial structures with a lateral resolution of 10 microm. The pressure-activated micro-syringe is equipped with a fine-bore exit needle using which a wide variety of 3D patterns with different arrays of channels (through-holes) were created. The channels can supply living cells with nutrients and allow removing the cell metabolites. The embedded cells remain viable and perform biological functions as long as the 3D structures are retained. The new technology has the potential for eventual high-throughput production of artificial human tissues and organs.

[Repair of the radial defect of rabbit by polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology].

OBJECTIVE: To evaluate the repairing effect of the rabbits radial defects of by polyester/tricalcium phosphate scaffolds prepared by rapid forming technology loaded with bovine BMP, and find a new carrier for growth factor. METHODS: Polyester/Tricalcium phosphate scaffolds prepared by rapid prototyping (RP) technology loaded with and without bovine BMP were used to repair the 15 mm radial defect of rabbit. Then results of radiography, histology, scaffolds degrade rates and bone density were appraised to examine the repairing effects of the scaffolds at 12 weeks. RESULTS: At 12 weeks, all defects treated with bBMP were radiographically repaired. No radii implanted polyester/tricalcium phosphate scaffolds alone showed radiographic and historical union. At experimental groups, longitudinal alignment of lamellar structure was observed histologically at 12 weeks, indicating that remodeling of regenerated bone almost completed, the scaffolds degradation rates were different by 12 weeks, and no abnormalities were observed in the surrounding soft tissue in all groups. CONCLUSION: Polyester/tricalcium phosphate scaffolds prepared by rapid prototyping technology loaded with bovine BMP can repair the rabbits radical defects. As for the effects, the poly (L-lactic-co-glycolide)/tricalcium phosphate (PLGA/TCP) scaffold are ideal and better than poly (L-lacide-co-D, L-lactide)/tricalcium phosphate (PDLLA/TCP) scaffold, but the poly (L-lactic acid)/tricalcium phosphate (PLLA/TCP) is not so good for its low degradation rates.

Effect of type I collagen on the adhesion, proliferation, and osteoblastic gene expression of bone marrow-derived mesenchymal stem cells.

OBJECTIVE: To investigate the effects of porous poly lactide-co-glycolide (PLGA) modified by type I collagen on the adhesion, proliferation, and differentiation of rabbit marrow-derived mesenchymal stem cells (MSCs). METHODS: The third generation MSCs isolated from mature rabbits by density gradient centrifugation were cultured at different initial concentrations on 0.3 cm x 1.2 cm x 2.0 cm 3-D porous PLGA coated by type I collagen in RPMI 1640 containing 10% fetal calf serum, while cultured on PLGA without type I collagen as control. The cells adhesive and proliferative behavior at 7, 14, and 21 days after inoculation was assessed by determining the incorporation rate of [(3)H]-TdR. In order to examine MSCs differentiation, the expression of osteoblasts marker genes, osteocalcin (OCN), alkaline phosphatase (ALP), osteopontin (OPN) mRNA, were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), and further more, the cell morphology at 21 days was also observed by scanning electron microscope (SEM). RESULTS: Type I collagen promoted cell adhesion on PLGA. The valve was significantly higher than controls (6 h, 2144 cpm+/-141 cpm vs. 1797 cpm+/-118 cpm, P=0.017; 8 h, 2311 cpm+/-113 cpm vs. 1891 cpm+/-103 cpm, P=0.01). The cells which cultured on PLGA coated with type I collagen showed significantly higher cell proliferation than controls on the 7 th day (1021 cpm+/-159 cpm vs. 451 cpm+/-67 cpm, P=0.002), the 14th day (1472 cpm+/-82 cpm vs. 583 cpm+/-67 cpm, P<0.001) and 21 th day (1728 cpm+/-78 cpm vs. 632 cpm+/-55 cpm, P<0.001). Osteoblasts markers, OCN, ALP, OPN mRNA, were all detected on PLGA coated by type I collagen on the 21 th day, but OCN, OPN mRNA could not be found in controls. Spindle and polygonal cells well distributed on the polymer coated by type I collagen while cylindric or round cells in controls. CONCLUSIONS: Type I collagen is effective in promoting the adhesion, proliferation and differentiation of MSCs on PLGA.

[Effects of collagen I coating on the porous poly-lactide-co-glycolid on adhesion, proliferation, and differentiation of mesenchymal stem cells].

OBJECTIVE: To investigate the effects of collagen I on the adhesion, proliferation, and differentiation of MSCs on PLGA. METHODS: Collagen I was added onto the surface of pores in pieces of 3-D porous poly-lactide-co-glycolid (PLGA). Bone marrow-derived mesenchymal stem cells (MSCs) were obtained from New Zealand rabbits and were cultured for 3 generations, inoculated into the pores of PLGA pieces with the volume of 0.3 cm x 1.2 cm x 2.0 cm, and then cultured in solution with [(3)H]-thymidine deoxyribose (TdR). PLGA pieces not coated by collagen I were used as controls. The incorporation rate of [(3)H]-TdR was detected 2, 4, 6, and 8 hours, and 7, 14, and 21 days after culture, shown in count per minute (CPM) value, to determine the adhesion and proliferation of the MSCs. RT-POCR was used to examine the expressions of mRNA of the osteoblast markers: osteocalcin (OCN), alkaline phosphatase (ALP), and osteopontin (OPN). Scanning electron microscopy (SEM) was used to observe the morphology of MSCs. RESULTS: The CPM value since 6 hours after culture between the experimental group and control group began to be significantly different (both P < 0.05) The CPM values 7, 14, and 21 days after culture between the experimental group and control groups (P < 0.05 or P < 0.01). OCN, ALP, and OPN mRNA were expressed in MSCs of the experimental group and only ALP mRNA was weakly expressed in the control group. SEM showed the distribution of spindle and polygonal cells in the pores of the 3-D PLGA pieces and distribution of cylindrical or round cells in the control group. CONCLUSION: Collagen I is effective in promoting the adhesion, proliferation, and differentiation of MSCs on PLGA.

[Rapid manufacturing of degradable porous polymer bone scaffold].

Making bone scaffold through tissue engineering method presents a new choice for both the patients and the doctors of orthopaedics. The biodegradable polymer PLA is chosen to make porous fundus scaffold jetting through special designed nozzle on multi-functional rapid prototyping machine controlled by computer according to the CT data CAD model. The scaffold is then chemically aggregated to compound with collagen-hydroxyapatite, and the ideal bone repair material is obtained. Animal experiment has indicated the correctness of this conclusion.

The impact of compact layer in biphasic scaffold on osteochondral tissue engineering.

The structure of an osteochondral biphasic scaffold is required to mimic native tissue, which owns a calcified layer associated with mechanical and separation function. The two phases of biphasic scaffold should possess efficient integration to provide chondrocytes and osteocytes with an independent living environment. In this study, a novel biphasic scaffold composed of a bony phase, chondral phase and compact layer was developed. The compact layer-free biphasic scaffold taken as control group was also fabricated. The purpose of current study was to evaluate the impact of the compact layer in the biphasic scaffold. Bony and chondral phases were seeded with autogeneic osteoblast- or chondrocyte-induced bone marrow stromal cells (BMSCs), respectively. The biphasic scaffolds-cells constructs were then implanted into osteochondral defects of rabbits' knees, and the regenerated osteochondral tissue was evaluated at 3 and 6 months after surgery. Anti-tensile and anti-shear properties of the compact layer-containing biphasic scaffold were significantly higher than those of the compact layer-free biphasic scaffold in vitro. Furthermore, in vivo studies revealed superior macroscopic scores, glycosaminoglycan (GAG) and collagen content, micro tomograph imaging results, and histological properties of regenerated tissue in the compact layer-containing biphasic scaffold compared to the control group. These results indicated that the compact layer could significantly enhance the biomechanical properties of biphasic scaffold in vitro and regeneration of osteochondral tissue in vivo, and thus represented a promising approach to osteochondral tissue engineering.

Skeletal repair in rabbits using a novel biomimetic composite based on adipose-derived stem cells encapsulated in collagen I gel with PLGA-beta-TCP scaffold.

In bone tissue engineering, the cell distribution mode in the scaffold may affect in vivo osteogenesis. Therefore, we fabricated a novel biomimetic construct based on a combination of rabbit adipose-derived stem cells (rASCs) encapsulated in collagen I gel with a PLGA-beta-TCP scaffold (rASCs-COL/PLGA-beta-TCP, group A), the combination of rASCs and PLGA-beta-TCP (rASCs/PLGA-beta-TCP, group B), the combination of collagen I gel and PLGA-beta-TCP (COL/PLGA-beta-TCP, group C), and PLGA-beta-TCP scaffold (group D). The composites were implanted into a 15-mm length critical-sized segmental radial defect. The results were assessed by histology, radiographs, bone mineral density (BMD), and mechanical testing. After 24 weeks, the medullary cavity recanalized, bone was rebuilt, and molding finished, the bone contour remodeled smoothly and the scaffold degraded completely in group A. The BMDs and mechanical properties were similar to normal. However, the bone defect remained unrepaired in groups B, C, and D. Moreover, the scaffold degradation rate in group A was significantly higher than the other groups. Thus, enhanced in vivo osteogenesis of rASCs wrapped in collagen I gel combined with PLGA-beta-TCP was achieved, and the bone defect was repaired. We hope this study provides new insights into ASCs-based bone tissue engineering.

Multinozzle low-temperature deposition system for construction of gradient tissue engineering scaffolds.

Tissue engineering is a technology that enables us to construct complicated hominine organs composed of many different types of cells. One of the key points to achieve this goal is to control the material composition and porous structure of the scaffold accurately. A disposable syringe based volume-driven injecting (VDI) nozzle was proposed and designed to extrude both natural derived and synthetic polymers. A multinozzle low-temperature deposition and manufacturing (M-LDM) system is proposed to fabricate scaffolds with heterogeneous materials and gradient hierarchical porous structures. PLGA, collagen, gelatin, chitosan can be extruded without leaking to form hierarchical porous scaffolds for primary study. Composite scaffolds with two kinds of materials were fabricated via two different nozzles to get both hydrophilic and mechanical properties. The results from scanning electron microscopy (SEM) demonstrated that the natural-derived biomaterials were strongly absorbed onto the synthetic biomaterials to form a stable network. Several gradient PLGA/TCP scaffolds were also fabricated to supply several samples.

Tissue anti-adhesion potential of biodegradable PELA electrospun membranes.

The most commonly used anti-adhesion device for separation and isolation of wounded tissues after surgery is the polymeric membrane. In this study, a new anti-adhesion membrane from polylactide-polyethylene glycol tri-block copolymer (PELA) has been synthesized. The synthesized copolymers were characterized by gel permeation chromatography and (1)H nuclear magnetic resonance spectroscopy. PELA membrane was prepared by electrospun. The prepared copolymer membranes were more flexible than the control poly-d-l-lactic acid (PDLLA) membrane, as investigated by the measurements of glass transition temperature. Its biocompatibility and anti-adhesion capabilities were also evaluated. In vitro cell adhesions on the PELA copolymer membrane and PDLLA membrane were compared by the culture of mouse fibroblasts L929 on the surfaces. For in vivo evaluation of tissue anti-adhesion potential, the PDLLA and PELA copolymer membranes were implanted between cecum and peritoneal wall defects of rats and their tissue adhesion extents were compared. It was observed that the PELA copolymer membrane was very effective in preventing cell or tissue adhesion on the membrane surface, probably owing to the effects of hydrophilic polyethylene glycol.

[Tissue engineered bone regeneration of periosteal cells using recombinant human bone morphogenetic protein 2 induce].

OBJECTIVE: To investigate bone regeneration of the cell-biomaterial complex using strategies of tissue engineering based on cells. METHODS: Hydroxyapatite/collagen (HAC) sandwich composite was produced to mimic the natural extracellular matrix of bone, with type I collagen serving as a template for apatite formation. A three-dimensional poly-porous scaffold was developed by mixing HAC with poly(L-lactic acid) (PLA) using a thermally induced phase separation technique (TIPS). The rabbit periosteal cells were treated with 500 ng/ml of recombinant human bone morphogenetic protein 2 (rhBMP-2), followed by seeded into pre-wet HAC-PLA scaffolds. Eighteen 3-month nude mice were implanted subcutaneously cell suspension (group A, n = 6), simple HAC-PLA scaffold (group B, n = 6) and cell-biomaterial complex (group C, n = 6) respectively. RESULTS: Using type I collagen to template mineralization of calcium and phosphate in solution, we get HAC sandwich composite, mimicking the natural bone both in composition and microstructure. The three dimensional HAC-PLA scaffold synthesized by TIPS had high porosity up to 90%, with pore size ranging from 50 microm to 300 microm. SEM examination proved that the scaffold supported the adhesion and proliferation of the periosteal cells. Histology results showed new bone formation 8 weeks after implantation in group C. The surface of group A was smooth without neoplasma. Fibrous tissue invasion occurred in group B and no bone and cartilage formations were observed. CONCLUSION: The constructed tissue engineering bone has emerged as another promising alternative for bone repair.