Evaluating 3D bone tissue engineered constructs with different seeding densities using the alamarBlue™ assay and the effect on in vivo bone formation

Bone tissue engineering using patient derived cells seeded onto porous scaffolds has gained much attention in recent years. Evaluating the viability of these 3D constructs is an essential step in optimizing the process. The alamarBlue (aB) assay was evaluated for its potential to follow in vitro cell proliferation on architecturally standardized hydroxyapatite scaffolds. The impact of the aB assayed and seeding density on subsequent in vivo bone formation was investigated. Twelve scaffolds were seeded with various densities from 250 to 2.5×10⁶ cells/scaffold and assay by aB at 5 time points during the 7-day culture period. Twelve additional scaffolds were seeded with 2.5×10⁵ cells/scaffold. Two control and 2 aB treated scaffolds were subcutaneously implanted into each of 6 nude mice for 6 weeks. Four observers ranked bone formation using a pair wise comparison of histological sections form each mouse. The aB assay successfully followed cell proliferation, however, the diffusion kinetics of the 3D constructs must be considered. The influence of in vitro aB treatment on subsequent in vivo bone formation cannot be ruled out but was not shown to be significant in the current study. The aB assay appears to be quite promising for evaluating a maximum or end-point viability of 3D tissue engineered constructs. Finally, higher seeding densities resulted in more observed bone formation.     

Alginate based scaffolds for bone tissue engineering

The design and production of scaffolds for bone tissue regeneration is yet unable to completely reproduce the native bone properties. In the present study new alginate microparticle and microfiber aggregated scaffolds were produced to be applied in this area of regenerative medicine.The scaffolds' mechanical properties were characterized by thermo mechanical assays. Their morphological characteristics were evaluated by isothermal nitrogen adsorption and scanning electron microscopy. The density of both types of scaffolds was determined by helium pycnometry and mercury intrusion porosimetry. Furthermore, scaffolds' cytotoxic profiles were evaluated in vitro by seeding human osteoblast cells in their presence.The results obtained showed that scaffolds have good mechanical and morphological properties compatible with their application as bone substitutes. Moreover, scaffold's biocompatibility was confirmed by the observation of cell adhesion and proliferation after 5 days of being seeded in their presence and by non-radioactive assays.     

Preparation of flexible bone tissue scaffold utilizing sea urchin test and collagen

Gonads of sea urchin are consumed in Japan and some countries as food and most parts including its tests are discarded as marine wastes. Therefore, utilization of them as functional materials would reduce the waste as well as encourage Japanese fishery. In this study, magnesium containing calcite granules collected from sea urchin tests were hydrothermally phosphatized and the obtained granules were identified as approximately 82% in mass of magnesium containing β-tricalcium phosphate and 18% in mass of nonstoichiometric hydroxyapatite, i.e., a biphasic calcium phosphate, maintaining the original porous network. Shape-controlled scaffolds were fabricated with the obtained biphasic calcium phosphate granules and collagen. The scaffolds showed good open porosity (83.84%) and adequate mechanical properties for handling during cell culture and subsequent operations. The MG-63 cells showed higher proliferation and osteogenic differentiation in comparison to a control material, the collagen sponge with the same size. Furthermore, cell viability assay proved that the scaffolds were not cytotoxic. These results suggest that scaffold prepared using sea urchin test derived calcium phosphate and collagen could be a potential candidate of bone void fillers for non-load bearing defects in bone reconstruction as well as scaffolds for bone tissue engineering.     

Migration of intervertebral disc cells into dense collagen scaffolds intended for functional replacement

Invasion of cells from surrounding tissues is a crucial step for regeneration when using a-cellular scaffolds as a replacement of the nucleus pulposus (NP). The aim of current study was to assess whether NP and surrounding annulus fibrosus (AF) cells are capable of migrating into dense collagen scaffolds. We seeded freshly harvested caprine NP and AF cells onto scaffolds consisting of 1.5 and 3.0% type I collagen matrices, prepared by plastic compression, to assess cell invasion. The migration distance appeared both time and density dependent and was higher for NP (25%) compared to AF (10%) cells after 4 weeks. Migration distance was not enhanced by Hst-2, a peptide derived from saliva known to enhance fibroblast migration, and this was confirmed in a scratch assay. In conclusion, we revealed invasion of cells into dense collagen scaffolds and therewith encouraging first steps towards the use of a-cellular scaffolds for NP replacement.     

Long-term viability of coronary artery smooth muscle cells on poly(L-lactide-co-epsilon-caprolactone) nanofibrous scaffold indicates its potential for blood vessel tissue engineering.

Biodegradable polymer nanofibres have been extensively studied as cell culture scaffolds in tissue engineering. However, long-term in vitro studies of cell-nanofibre interactions were rarely reported and successful organ regeneration using tissue engineering techniques may take months (e.g. blood vessel tissue engineering). Understanding the long-term interaction between cells and nanofibrous scaffolds (NFS) is crucial in material selection, design and processing of the tissue engineering scaffolds. In this study, poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] (70:30) copolymer NFS were produced by electrospinning. Porcine coronary artery smooth muscle cells (PCASMCs) were seeded and cultured on the scaffold to evaluate cell-nanofibre interactions for up to 105 days. A favourable interaction between this scaffold and PCASMCs was demonstrated by cell viability assay, scanning electron microscopy, histological staining and extracellular matrix (ECM) secretion. Degradation behaviours of the scaffolds with or without PCASMC culture were determined by mechanical testing and gel permeation chromatography (GPC). The results showed that the PCASMCs attached and proliferated well on the P(LLA-CL) NFS. Large amount of ECM protein secretion was observed after 50 days of culture. Multilayers of aligned oriented PCASMCs were formed on the scaffold after two months of in vitro culture. In the degradation study, the PCASMCs were not shown to significantly increase the degradation rate of the scaffolds for up to 105 days of culture. The in vitro degradation time of the scaffold could be as long as eight months by extrapolating the results from GPC. These observations further supported the potential use of the P(LLA-CL) nanofibre in blood vessel tissue engineering.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.     

Enhanced physicochemical properties of collagen by using EDC/NHS-crosslinking

Collagen-based scaffolds are appealing products for the repair of cartilage defects using tissue engineering strategies. The present study investigated the collagen scaffolds with and without 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)-crosslinking. Crosslinking density, matrix morphology, swelling ratio shrinkage temperature and resistance against collagenase digestion were determined to evaluate the physicochemical properties of the collagen matrices with and without crosslinking. The results conformed that the porous structure of collagen was largely preserved and adjusted by crosslinking treatment. Furthermore, crosslinked collagen samples showed significantly reduced swelling ratio and increased resistance against thermal treatment and enzymatic degradation compared to non-crosslinked samples. An in vitro evaluation of MC3T3-E1 cells seeded onto the crosslinked and non-crosslinked collagen matrix indicated that crosslinked collagen was nontoxic and improved cell proliferation. Through this work, it was shown that an osteoconductive collagen matrix with optimized properties used as bioactive and bioresorbable scaffolds in bone tissue engineering could be fabricated through the EDC/NHS-crosslinking method.     

Control of morphological properties of porous biodegradable scaffolds processed by supercritical CO2 foaming

A low cost supercritical CO₂ foaming rig with a novel design has been used to prepare fully interconnected and highly porous biodegradable scaffolds with controllable pore size and structure that can promote cancellous bone regeneration. Porous polymer scaffolds have been produced by plasticising the polymer with high pressure CO₂ and by the formation of a porous structure following the escape of CO₂ from the polymer. Although, control over pore size and structure has been previously reported as difficult with this process, the current study shows that control is possible. The effects of processing parameters such as CO₂ saturation pressure, time and temperature and depressurisation rate on the morphological properties, namely porosity, pore interconnectivity, pore size and wall thickness- of the scaffolds have been investigated. Poly(d,l)lactic acid was used as the biodegradable polymer. The surfaces and internal morphologies of the poly(d,l)lactic acid scaffolds were examined using optical microscope and micro computed tomography. Preosteoblast human bone cells were seeded on the porous scaffolds in vitro to assess cell attachment and viability. The scaffolds showed a good support for cell attachment, and maintained cell viability throughout 7 days in culture. This study demonstrated that the morphology of the porous structure can be controlled by varying the foaming conditions, allowing the porous scaffolds to be used in various tissue engineering applications.     

Human-like collagen/nano-hydroxyapatite scaffolds for the culture of chondrocytes

Three dimensional (3D) biodegradable porous scaffolds play a key role in cartilage tissue repair. Freeze-drying and cross-linking techniques were used to fabricate a 3D composite scaffold that combined the excellent biological characteristics of human-like collagen (HLC) and the outstanding mechanical properties of nano-hydroxyapatite (nHA). The scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and compression tests, using Relive® Artificial Bone (RAB) scaffolds as a control. HLC/nHA scaffolds displayed homogeneous interconnected macroporous structure and could withstand a compression stress of 2.67 ± 0.37 MPa, which was higher than that of the control group. Rabbit chondrocytes were seeded on the composite porous scaffolds and cultured for 21 days. Cell/scaffold constructs were examined using SEM, histological procedures, and biochemical assays for cell proliferation and the production of glycosaminoglycans (GAGs). The results indicated that HLC/nHA porous scaffolds were capable of encouraging cell adhesion, homogeneous distribution and abundant GAG synthesis, and maintaining natural chondrocyte morphology compared to RAB scaffolds. In conclusion, the presented data warrants the further exploration of HLC/nHA scaffolds as a potential biomimetic platform for chondrocytes in cartilage tissue engineering.     

Biological response to pre-mineralized starch based scaffolds for bone tissue engineering

It is known that calcium-phosphate (Ca-P) coatings are able not only to improve the bone bonding behaviour of polymeric materials, but at the same time play a positive role on enhancing cell adhesion and inducing the differentiation of osteoprogenitor cells. Recently an innovative biomimetic methodology, in which a sodium silicate gel was used as a nucleative agent, was proposed as an alternative to the currently available biomimetic coating methodologies. This methodology is especially adequate for coating biodegradable porous scaffolds. In the present work we evaluated the influence of the referred to treatment on the mechanical properties of 50/50 (wt%) blend of corn starch/ethylene-vinyl alcohol (SEVA-C) based scaffolds. These Ca-P coated scaffolds presented a compressive modulus of 224.6 ± 20.6 and a compressive strength of 24.2 ± 2.20. Cytotoxicity evaluation was performed according ISO/EN 10993 part 5 guidelines and showed that the biomimetic treatment did not have any deleterious effect on L929 cells and did not inhibit cell growth. Direct contact assays were done by using a cell line of human osteoblast like cells (SaOS-2). 3 × 10⁵ cells were seeded per scaffold and allowed to grow for two weeks at 37C in a humidified atmosphere containing 5% CO₂. Total protein quantification and scanning electron microscopy (SEM) observation showed that cells were able to grow in the pre-mineralized scaffolds. Furthermore cell viability assays (MTS test) also show that cells remain viable after two weeks in culture. Finally, protein expression studies showed that after two weeks osteopontin and collagen type I were being expressed by SaOS-2 cells seeded on the pre-mineralized scaffolds. Moreover, alkaline phosphatase (ALP) activity was higher in the supernatants collected from the pre-mineralized samples, when compared to the control samples (non Ca-P coated). This may indicate that a faster mineralization of the ECM produced on the pre-mineralized samples was occurring. Consequently, biomimetic pre-mineralization of starch based scaffolds can be a useful route for applying these materials on bone tissue engineering.     

Facile fabrication of the glutaraldehyde cross-linked collagen/chitosan porous scaffold for skin tissue engineering

Porous scaffold is one of the key factors in skin tissue engineering. In this study, a facile method was developed to prepare the glutaraldehyde (GA) cross-linked collagen/chitosan porous scaffold (S2). The properties of S2 were compared with the scaffolds prepared by the traditional method (S1). Compared to the rough surface and collapsed inner structure of S1, S2 showed a smooth surface and controlled size. After treated by GA with same concentration, S1 and S2 showed the similar swelling ratios, which are big enough to ensure the nutrient supply in the early stage of wound healing. The effects of the fabrication methods as well as the GA concentration on the cross-linking degree and in vitro degradation degree of the scaffolds were studied. It was found that the cross-linking degree of S2-0.25% was much higher than that of S1. Investigation of the tensile and compression properties of the scaffolds found that the mechanical property of S2-0.04% is closest to that of S1. High performance liquid chromatography (HPLC) was applied to determine the residual GA. The results proved that, compared to water rinse, oven drying is a feasible and effective method to remove the residual GA. Finally, the cytocompatibility of S2 was evaluated by in vitro culture of fibroblasts. The results of cell morphology and cell viability proved that S2-0.04% could retain the original good cytocompatibility of S1 to accelerate cell infiltration and proliferation effectively. All these results indicate that it is a feasible method to prepare the GA cross-linked collagen/chitosan scaffold.     

Surface modification of three-dimensional Ca-P/PHBV nanocomposite scaffolds by physical entrapment of gelatin and its in vitro biological evaluation

The properties of bone tissue engineering scaffolds such as architecture, porosity, mechanical properties and surface properties have significant effects on cellular response and play an important role in bone regeneration. In this study, three-dimensional nanocomposite scaffolds consisting of calcium phosphate (Ca-P) nanoparticles and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) copolymer with controlled external and internal architectures were successfully produced via selective laser sintering (SLS), one of the versatile rapid prototyping techniques. The Ca-P/PHBV nanocomposite scaffolds had a porosity of (61.75±1.24)%, compressive strength of (2.16±0.21) MPa and Young’s modulus of (26.98±2.29) MPa. The surface modification of scaffolds by gelatin was achieved through physical entrapment. The amount of entrapped gelatin could be controlled by varying the solvent composition and reaction time. The surface modification improved the hydrophilicity of scaffolds but did not significantly affect the surface morphology and mechanical properties. Osteoblast-like cells (SaOS-2) were cultured on scaffolds with and without gelatin surface modification. The majority of SaOS-2 cells were viable and proliferated in both types of scaffolds for up to 14 d in culture, as indicated by MTT assay and live and dead assay. Surface modification significantly increased cell proliferation for surface modified scaffolds, which could be due to the improvement in hydrophilicity of the scaffolds.     

Sol–gel derived composite from bioactive glass–polyvinyl alcohol

Investigation of novel biomaterials for bone engineering is based on the development of porous scaffolds, which should match the properties of the tissue that is to be replaced. These materials need to be biocompatible, ideally osteoinductive, osteoconductive, and mechanically well-matched. In the present paper, we report the preparation and characterization of hybrid macroporous scaffold of polyvinyl alcohol (PVA)/bioactive glass through the sol–gel route. Hybrids containing PVA (80, 70 and 60 wt%) and bioactive glass with composition 58SiO₂–33CaO–9P₂O₅ were synthesized by foaming a mixture of polymer solution and bioactive glass via sol–gel precursor solution. PVA with two different degree of hydrolysis (DH), 98.5% (high degree) and 80% (low degree) were also investigated, in order to evaluate the influence of residual acetate group present in polymer chain on the final structure and properties of 3D porous composite produced. The microstructure, morphology and crystallinity of the hybrid porous scaffolds were characterized by X-ray diffraction (XRD), Infrared Fourier Transform spectrometry (FTIR) and Scanning electron microscopy (SEM/EDX) analysis. In addition, specific surface area was assessed by B.E.T. nitrogen adsorption method and mechanical behavior was evaluated by compression tests. Preliminary cytotoxicity and cell viability were also performed by the MTT assay. VERO cell monolayers were grown in 96-well microtiter plates. The results have clearly showed that hybrid foams of polyvinyl alcohol/bioactive glass (PVA/BG) with interconnected macroporous 3D structure were successfully produced. All the tested hybrids of PVA/BG have showed adequate cell viability properties for potential biological applications.     

Morphological,mechanical, and biocompatibility characterization of macroporous alumina scaffolds coated with calcium phosphate/PVA

In bone tissue engineering, a highly porous artificial extracellular matrix or scaffold is required to accommodate cells and guide the tissue regeneration in three-dimension. Calcium phosphate (CaP) ceramics are widely used for bone substitution and repair due to their biocompatibility, bioactivity, and osteoconduction. However, compared to alumina ceramics, either in the dense or porous form, the mechanical strength achieved for calcium phosphates is generally lower. In the present work, the major goal was to develop a tri-dimensional macroporous alumina scaffold with a biocompatible PVA/calcium phosphate coating to be potentially used as bone tissue substitute. This approach aims to combine the high mechanical strength of the alumina scaffold with the biocompatibility of calcium phosphate based materials. Hence, the porous alumina scaffolds were produced by the polymer foam replication procedure. Then, these scaffolds were submitted to two different coating methods: the biomimetic and the immersion in a calcium phosphate/polyvinyl alcohol (CaP/PVA) slurry. The microstructure, morphology and crystallinity of the macroporous alumina scaffolds samples and coated with CaP/PVA were characterized by X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM/EDX) analysis. Also, specific surface area was assessed by BET nitrogen adsorption method and mechanical behavior was evaluated by axial compression tests. Finally, biocompatibility and cytotoxicity were evaluated by VERO cell spreading and attachment assays under SEM. The morphological analysis obtained from SEM photomicrograph results has indicated that 3D macroporous alumina scaffolds were successfully produced, with estimated porosity of over 65% in a highly interconnected network. In addition, the mechanical test results have indicated that porous alumina scaffolds with ultimate compressive strength of over 3.0 MPa were produced. Concerning to the calcium phosphate coatings, the results have showed that the biomimetic method was not efficient on producing a detectable layer onto the alumina scaffolds. On the other hand, a uniform and adherent inorganic–organic coating was effectively formed onto alumina macroporous scaffold by the immersion of the porous structure into the CaP/PVA suspension. Viable VERO cells were verified onto the surface of alumina porous scaffold samples coated with PVA–calcium phosphate. In conclusion, a new method was developed to produce alumina with tri-dimensional porous structure and uniformly covered with a biocompatible coating of calcium phosphate/PVA. Such system has high potential to be used in bone tissue engineering.     

Biofabrication and in vitro study of hydroxyapatite/mPEG–PCL–mPEG scaffolds for bone tissue engineering using air pressure-aided deposition technology

The aims of this study were to fabricate biopolymer and biocomposite scaffolds for bone tissue engineering by an air pressure-aided deposition system and to carry out osteoblast cell culture tests to validate the biocompatibility of fabricated scaffolds. A mPEG–PCL–mPEG triblock copolymer was synthesized as a biopolymer material. Biocomposite material was composed of synthesized biopolymer and hydroxyapatite (HA) with a mean diameter of 100 μm. The weight ratio of HA added to the synthesized biopolymer was 0.1, 0.25, 0.5 and 1. The experimental results show that the maximum average compressive strength of biocomposite scaffolds, made of weight ratio 0.5, with mean pore size of 410 μm (porosity 81%) is 18.38 MPa which is two times stronger than that of biopolymer scaffolds. Osteoblast cells, MC3T3-E1, were seeded on both types of fabricated scaffolds to validate the biocompatibility using methylthianzol tetrazolium (MTT) assay and cell morphology observation. After 28 days of in vitro culturing, the seeded osteoblasts were well distributed in the interior of both types of scaffolds. Furthermore, MTT experimental results show that the cell viability of the biocomposite scaffold is higher than that of the biopolymer scaffold. This indicates that adding HA into synthesized biopolymer can enhance compressive strength and the proliferation of the osteoblast cell.     

Influencing biophysical properties of fibrin with buffer solutions

Fibrin has been proposed as cell scaffold for numerous tissue engineering applications. While most of the studies have focused on fibrinogen and thrombin, other components of fibrin can also affect its properties. The present study aimed to evaluate the effects of buffer solution composition on fibrin biophysical properties. Fibrin scaffolds were synthesized with different calcium, chloride, and factor XIII (FXIII) final concentrations. Light transmission was determined as a relative, semi-quantitative estimator of fiber structure differences, and two compositions, resulting in translucent and opaque gels, were tested for mechanical and biological properties. Gels were seeded with mouse mesenchymal cells, C3H10T1/2, or bovine bone marrow-derived mesenchymal stromal cells and cultured up to 10 or 24 days, before cell number, morphology and distribution were evaluated. Calcium increased gel opacity (i.e., fiber thickness), while chloride and FXIII decreased it. Opaque gels displayed a fluid-like viscous behavior while translucent gels showed improved elastic properties. Both compositions supported survival of both cell types with opaque gels leading to better proliferation, but significant scaffold shrinkage after 17 days of culture. These results demonstrated that calcium, chloride, and FXIII modulate the biophysical properties of fibrin, and can be used to adjust mechanical and biological properties for tissue engineering applications.     

Static and dynamic cultivation of bone marrow stromal cells on biphasic calcium phosphate scaffolds derived from an indirect rapid prototyping technique

The adequate regeneration of large bone defects is still a major problem in orthopaedic surgery. Synthetic bone substitute materials have to be biocompatible, biodegradable, osteoconductive and processable into macroporous scaffolds tailored to the patient specific defect. Hydroxyapatite (HA) and tricalcium phosphate (TCP) as well as mixtures of both phases, biphasic calcium phosphate ceramics (BCP), meet all these requirements and are considered to be optimal synthetic bone substitute materials. Rapid prototyping (RP) can be applied to manufacture scaffolds, meeting the criteria required to ensure bone ingrowth such as high porosity and defined pore characteristics. Such scaffolds can be used for bone tissue engineering (BTE), a concept based on the cultivation of osteogenic cells on osteoconductive scaffolds. In this study, scaffolds with interconnecting macroporosity were manufactured from HA, TCP and BCP (60 wt% HA) using an indirect rapid prototyping technique involving wax ink-jet printing. ST-2 bone marrow stromal cells (BMSCs) were seeded onto the scaffolds and cultivated for 17 days under either static or dynamic culture conditions and osteogenic stimulation. While cell number within the scaffold pore system decreased in case of static conditions, dynamic cultivation allowed homogeneous cell growth even within deep pores of large (1,440 mm³) scaffolds. Osteogenic cell differentiation was most advanced on BCP scaffolds in both culture systems, while cells cultured under perfusion conditions were generally more differentiated after 17 days. Therefore, scaffolds manufactured from BCP ceramic and seeded with BMSCs using a dynamic culture system are the method of choice for bone tissue engineering.     

Tenocyte proliferation on collagen scaffolds protects against degradation and improves scaffold properties

Tissue engineering scaffolds encourage cell proliferation whilst degrading to facilitate tissue regeneration. Their mechanical properties therefore change, decreasing due to scaffold degradation and increasing due to extracellular matrix deposition. This work compares the changing properties of collagen scaffolds incubated in culture medium, with and without human tenocytes, in order to investigate the relationship between degradation and tenocyte proliferation. The material properties of scaffolds are compared over 26 days using mechanical testing, differential scanning calorimetry, infra-red spectroscopy, and histology and biochemical assays. For medium-only scaffolds, the mechanical properties decrease rapidly, while culture medium sulfhydryl content increases significantly, with no significant changes in the denaturation temperature of scaffold collagen content. Conversely, the mechanical properties and collagen content of tenocyte-seeded scaffolds increase significantly while culture medium sulfhydryl content decreases and denaturation temperature remains the same. These results indicate that tenocytes proliferation both reduces the degradation of collagen scaffolds incubated in culture medium and produces scaffolds with improved properties.     

Chitosan/poly(dl,lactide-co-glycolide) scaffolds for tissue engineering

Chitosan/poly(dl-lactide-co-glycolide) (Ch/dl PLG) composite scaffolds were fabricated by freeze-drying lyophilization, and were evaluated and compared for use as a bone regeneration scaffold through measurements of the compression mechanical properties of the porous scaffolds. Also, In vitro cell culture of Sprague?CDawley rat??s osteoblasts were used to evaluate the phenotype expression of cells in the scaffolds, characterizing the cellular adhesion, proliferation and alkaline phosphatase activity. The gene expression of osteocalcin, sialoprotein, alkaline phosphatase, Type I collagen and TGF??1 were confirmed in the samples; moreover, it was confirmed, the mineralization by IR spectra and EDS analysis. Our results thus show that Ch/dl PLG scaffolds are suitable for biological applications.     

Synthesis and characterization of calcium phosphate/collagen biocomposites doped with Zn2+

Composites were developed using calcium phosphate (CaP)/collagen (COL) doped with Zn⁺² to attempt the materials association with adequate properties for biological applications in the recovery of the bone tissue by trauma or pathogenies. Hydroxyapatite (HAP) and hydroxyapatite-βtricalcium phosphate (HAPβTCP) were synthesized and doped with zinc nitrate. High purity grade type I collagen was extracted and purified from bovine pericardium. CaP doped and undoped with Zn⁺² were produced with COL and the composites were developed using a simple mixture process. All samples were characterized by Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR) and X-ray diffraction analysis (XRD. In addition, biocompatibility and cell viability were assessed by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) using osteoblast cell culture. The results have indicated that both morphological and structural features and chemical composition of the composites were very similar to their precursors, collagen and calcium phosphate components. Also, the biocomposites presented a homogeneous aspect with the calcium phosphate particles aggregated to the collagen fibers. The biological evaluation of the composites in vitro showed cellular viability, presenting proliferation of the osteoblasts compared to the control cells (P < 0.05). The composites showed appropriate physical and biological properties creating more biologically active scaffolds that may support bone growth. Therefore, the novel developed biocomposites have high potential to be used for rebuilding small lesions in bone tissue engineering.