Mineralised collagen—an artificial,extracellular bone matrix—improves osteogenic differentiation of bone marrow stromal cells

In the field of bone tissue engineering there is a high demand on bone graft materials which promote bone formation. By combination of collagen type I with nanocrystalline hydroxyapatite (HA) we generated a resorbable material which structure and composition is close to those of the extracellular bone matrix. This nanocomposit material was produced in a biomimetic process in which collagen fibril assembly and mineralisation with hydroxyapatite occur simultaneously. In this study the proliferation and osteogenic differentiation of human bone marrow-derived stromal cells (hBMSC) on membranes of biomimetically mineralised collagen type I was investigated. To this end, we optimised biochemical assays for determination of cell number and alkaline phosphatase activity corresponding to the special properties of this biomaterial. For cell experiments hBMSC were seeded on the mineralised collagen membranes and cultivated over 35 days, both in static and perfusion culture, in the presence and absence of dexamethasone, β-glycerophosphate and ascorbate. Compared to cells grown on tissue culture polystyrene we found attenuated proliferation rates, but markedly increased activity of alkaline phosphatase on the mineralised collagen indicating its promoting effect on the osteogenic differentiation of hBMSC. Therefore this bone-like material may act as a suitable artificial extracellular matrix for bone tissue engineering. Perfusion of the 2D cell matrix constructs with cell culture medium did not improve proliferation and osteogenic differentiation of the hBMSC. Anne Bernhardt and Anja Lode contributed equally to this paper     

The fundamental parameters of chitosan in polymer scaffolds affecting osteoblasts (MC3T3-E1)

The aim of this study was to investigate the degree of deacetylation (DD) and molecular weight (MW) of chitosan within chitosan–collagen scaffolds on mouse osteoblasts (MC3T3-E1). The chitosan–collagen scaffolds were fabricated by freeze-drying technique. The studies on cell attachment and proliferation, alkaline phosphatase (ALP) activity, cell morphology, and mineralized nodule formation by osteoblasts on scaffolds were investigated. No statistically significant difference was found on cell attachment, but the chitosan–collagen scaffolds with low-DD chitosan had a statistically significantly (P < 0.05) higher proliferative effect and ALP activity than those scaffolds with high-DD chitosan, regardless of molecular weight. Scanning electron images demonstrated that MC3T3-E1 cells grew well on all test scaffolds; on the contrary, mineralized nodule formation was not found. In conclusion, the DD of chitosan is a crucial factor for MC3T3-E1 cells and it should be considered in further applications for bone tissue engineering.     

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(ε-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA–PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA–PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA–PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA–PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering. Seok-Jung Hong and Ishik Jeong contributed equally.     

Poröse Scaffolds aus mineralisiertem Kollagen – ein biomimetisches Knochenersatzmaterial

Porous scaffolds made from mineralised collagen – a biomimetic bone graft material Using biomimetically mineralised collagen type I, a new porous bone graft material has been developed, the composition of which mimics extracellular matrix of bone tissue. The pore structure is generated by a freeze drying process, whereas the pore size can be controlled by temperature and velocity of the freezing over a wide range. The structure is stabilized by crosslinking of the collagen with the water soluble carbodiimide derivative EDC. For the seeding with bone cells, pores with diameters of about 200 μm have turned out to be optimal. These scaffolds are elastic in the wet state which allow for cell culture experiments under mechanical stimulation.     

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.     

Wet chemical process to enhance osteoconductivity of electrospun chitosan nanofibers

In this study, chitosan/hydroxyapatite (CS/HA) nanofibers were prepared using a wet chemical method. First, CS nanofibers with uniform diameters were fabricated using electrospinning. Then, a wet chemical process was used to mineralize nanofiber surfaces to form a homogeneous HA deposit. Reactions with three cycles were found to optimize biomimetic properties of the HA. The mineralization process required only approximately 3 h, which corresponded to a saving of 98 % in preparation time compared with that needed by the process using a simulated body fluid (SBF). According to the attachment and spreading of UMR (rat osteosarcoma) cells on the CS/HA composite fibers, the deposited mineralization layer significantly enhanced cell affinity of the CS nanofibers and the HA created by the wet chemical method was as effective as that created by the SBF. The composite nanofibrous scaffolds produced by the wet chemical process also promoted osteogenic differentiation by inducing ossification. Thus, expressions of collagen type I, alkaline phosphatase, osteocalcin, bone sialoprotein, and osterix were all enhanced. These results demonstrated that composite electrospun fibers can be efficiently prepared using wet chemical method and the resulting nanofibrous scaffolds have considerable potential in future bone tissue engineering applications.     

Primary osteoblast cell response to sol-gel derived bioactive glass foams

Bioactive glass macroporous structures were developed in this work to be used as scaffolds for bone tissue engineering applications. A sol-gel route was used to obtain glass foams with the introduction of a gas phase in the solution and by vigorous agitation of the sol-gel solution that contains a foam agent. Stable and homogeneous foams were formed near the gelation point, which were than dried and heat-treated. Macroporous structures with interconnected pores of up to 500 μ m, porosity as high as 88% and specific surface area of 92 m²/g were obtained. The porous glasses were tested in osteoblast cultures to evaluate adhesion, proliferation, collagen and alkaline phosphatase production. Osteoblast proliferation was higher in the presence of the foams as well as was the collagen secretion, when compared to control. The alkaline phosphatase production was not altered. Viable osteoblasts could be seen inside the foams, suggesting that the produced porous glass foams are a promising materials for bone repair, since it provides a good environment for the adhesion and proliferation of osteoblasts.     

Cell response to collagen-calcium phosphate cement scaffolds investigated for nonviral gene delivery

Collagen-hydroxyapatite (HA) scaffolds for the non-viral delivery of a plasmid encoding the osteoinductive protein bone morphogenetic protein (BMP)-7 were developed. The collagen-HA was obtained by the combination of calcium phosphate cement in a collagen template. The effect on cell behavior of increasing amounts of HA in the scaffolds was evaluated. Collagen-HA scaffolds containing 13, 23 or 83 wt% HA were prepared. Cell proliferation was reduced in the 83% HA scaffold after 1 day compared to 13 and 23% HA, but by 14 days the number of cells in 83% HA considerably increased. Alkaline phosphatase (ALP) activity was 8 times higher for the 83% HA scaffolds. BMP-7 plasmid was incorporated into the 83% HA scaffold. The transfection was low, although significant levels of BMP7 were expressed, associated with an increase in cell proliferation.     

Biological characteristic effects of human dental pulp stem cells on poly-ε-caprolactone-biphasic calcium phosphate fabricated scaffolds using modified melt stretching and multilayer deposition

Craniofacial bone defects such as alveolar cleft affect the esthetics and functions that need bone reconstruction. The advanced techniques of biomaterials combined with stem cells have been a challenging role for maxillofacial surgeons and scientists. PCL-coated biphasic calcium phosphate (PCL-BCP) scaffolds were created with the modified melt stretching and multilayer deposition (mMSMD) technique and merged with human dental pulp stem cells (hDPSCs) to fulfill the component of tissue engineering for bone substitution. In the present study, the objective was to test the biocompatibility and biofunctionalities that included cell proliferation, cell viability, alkaline phosphatase activity, osteocalcin, alizarin red staining for mineralization, and histological analysis. The results showed that mMSMD PCL-BCP scaffolds were suitable for hDPSCs viability since the cells attached and spread onto the scaffold. Furthermore, the constructs of induced hDPSCs and scaffolds performed ALP activity and produced osteocalcin and mineralized nodules. The results indicated that mMSMD PCL-BCP scaffolds with hDPSCs showed promise in bone regeneration for treatment of osseous defects.     

Enhanced osteogenic differentiation of cord blood-derived unrestricted somatic stem cells on electrospun nanofibers

A new stem cell-scaffold construct based on poly-l-lactide (PLLA) nanofibers grafted with collagen (PLLA-COL) and cord blood-derived unrestricted somatic stem cells (USSC) were proposed to hold promising characteristics for bone tissue engineering. Fabricated nanofibers were characterized using SEM, ATR-FTIR, tensile and contact angle measurements. The capacity of PLLA, plasma-treated PLLA (PLLA-pl) and PLLA-COL scaffolds to support proliferation and osteogenic differentiation of USSC was evaluated using MTT assay and common osteogenic markers such as alkaline phosphatase (ALP) activity, calcium mineral deposition and bone-related genes. All three scaffolds showed nanofibrous and porous structure with suitable physical characteristics. Higher proliferation and viability of USSC was observed on PLLA-COL nanofibers compared to control surfaces. In osteogenic medium, ALP activity and calcium deposition exhibited the highest values on PLLA-COL scaffolds on days 7 and 14. These markers were also greater on PLLA and PLLA-pl compared to TCPS. Higher levels of collagen I, osteonectin and bone morphogenetic protein-2 were detected on PLLA-COL compared to PLLA and PLLA-pl. Runx2 and osteocalcin were also expressed continuously on all scaffolds during induction. These observations suggested the enhanced proliferation and osteogenic differentiation of USSC on PLLA-COL nanofiber scaffolds and introduced a new combination of stem cell-scaffold constructs with desired characteristics for application in bone tissue engineering.     

Hardystonite improves biocompatibility and strength of electrospun polycaprolactone nanofibers over hydroxyapatite: A comparative study

The aim of this study was to compare physico-chemical and biological properties of hydroxyapatite (HA) and hardystonite (HS) based composite scaffolds. Hardystonite (Ca₂ZnSi₂O₇) powders were synthesized by a sol–gel method while polycaprolactone–hardystonite (PCL–HS) and polycaprolactone–hydroxyapatite (PCL–HA) were fabricated in nanofibrous form by electrospinning. The physico-chemical and biological properties such as tensile strength, cell proliferation, cell infiltration and alkaline phosphatase activity were determined on both kinds of scaffolds. We found that PCL–HS scaffolds had better mechanical strength compared to PCL–HA scaffolds. Addition of HA and HS particles to PCL did not show any inhibitory effect on blood biocompatibility of scaffolds when assessed by hemolysis assay. The in vitro cellular behavior was evaluated by growing murine adipose-tissue-derived stem cells (mE-ASCs) over the scaffolds. Enhanced cell proliferation and improved cellular infiltrations on PCL–HS scaffolds were observed when compared to HA containing scaffolds. PCL–HS scaffolds exhibited a significant increase in alkaline phosphatase (ALP) activity and better mineralization of the matrix in comparison to PCL–HA scaffolds. These results clearly demonstrate the stimulatory role of Zn and Si present in HS based composite scaffolds, suggesting their potential application for bone tissue engineering.     

In vitro behavior of osteoblast-like cells on PLLA films with a biomimetic apatite or apatite/collagen composite coating

To investigate the methods to improve the cell–material interaction of devices or tissue engineering scaffolds made of poly(l-lactic acid) (PLLA) polymer, apatite and apatite/collagen composite coatings were formed on PLLA films within 24 h through accelerated biomimetic processes. In vitro investigation using Saos-2 osteoblast-like cells through cell culture was conducted to assess the biological performance of these biomimetic coatings. The cell morphology on three types of surfaces, viz., PLLA film, PLLA film with the apatite coating, and PLLA film with the apatite/collagen composite coating, was studied using scanning electron microscopy (SEM). Cell viability was estimated using the MTT assay. The differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. The results obtained indicated that the biomimetic apatite and apatite/collagen composite coatings could significantly enhance the proliferation and differentiation of osteoblast-like cells. The apatite/collagen composite coating appears to be promising for the surface modification of PLLA-based devices with much improved interactions with osteoblastic cells.     

Providing osteogenesis conditions to mesenchymal stem cells using bioactive nanocomposite bone scaffolds

Development of bone scaffolds with excellent osteogenic potential is highly important for stem cell-based bone engineering. Here we developed novel scaffolds made of poly(lactic acid) (PLA) biopolymer with bioactive glass nanocomponent. In vitro bone bioactivity and osteogenic potential of the nanocomposite scaffolds were determined using bone marrow mesenchymal stem cells. Glass nanocomponent was evenly embedded within the PLA matrix while preserving the scaffold pore structure. Simulated body fluid (SBF) test revealed rapid induction of bone mineral-like apatite over the surface of the nanocomposite scaffold, which was not readily observed in the PLA. Cells adhered well onto the nanocomposite scaffold and multiplied during culture period. Nanocomposite scaffold significantly stimulated alkaline phosphatase (ALP) activity and the expression of bone-associated genes (collagen I, ALP, osteopontin and osteocalcin) with respect to PLA. Western blot analysis confirmed the osteogenic protein level was also higher on the nanocomposite scaffold. Results suggest that the nanocomposite scaffolds provide favorable conditions for osteogenesis of MSCs and thus find potential uses in bone tissue engineering.     

Fabrication of biocompatible titanium scaffolds using space holder technique

Open-pore titanium scaffolds were fabricated by sintering of compressed mixtures of TiH_1.924 and urea. Spherical and irregular shaped space holders were used to investigate the effect of pore shape on cellular behavior. After removal of the space holder, the shape of the spacers was replicated to the pores. Average diameter of the pores was in the range of 300–600?μm. SEM images showed that titanium hydride resulted in higher surface roughness and larger micro porosities than pure titanium. In vitro evaluations were carried out by using MTT assay, measuring alkaline phosphatase activity and alizarin red staining in flow perfusion bioreactor for cell culture. Observations revealed excellent attachment and proliferation of G-292 cells to the highly porous scaffolds fabricated with titanium hydride and urea of this research.     

Local delivery of alendronate eluting chitosan scaffold can effectively increase osteoblast functions and inhibit osteoclast differentiation

The aim of this study was to investigate the effect of alendronate released from chitosan scaffolds on enhancement of osteoblast functions and inhibition of osteoclast differentiation in vitro. The surface and cell morphologies of chitosan scaffolds and alendronate-loaded chitosan scaffolds were characterized by variable pressure field emission scanning electron microscope (VP-FE-SEM). Alendronate was released in a sustained manner. For evaluating osteoblast functions in MG-63 cells, we investigated cell proliferation, alkaline phosphatase (ALP) activity, and calcium deposition. Furthermore, for evaluating inhibition of osteoclast differentiation in RAW 264.7 cells, we investigated tartrate-resistant acid phosphatase (TRAP) activity, TRAP staining, and gene expressions. The in vitro studies revealed that osteoblasts grown on alendronate-loaded chitosan scaffold showed a significant increment in cell proliferation, ALP activity, and calcium deposition as compared to those grown on chitosan scaffolds. In addition, the in vitro study showed that osteoclast differentiation in RAW 264.7 cells cultured on alendronate-loaded chitosan scaffolds was greatly inhibited as compared to those cultured on chitosan scaffolds by the results of TRAP activity, TRAP staining, and gene expressions. Taken together, alendronate-loaded chitosan scaffolds could achieve the dual functions of improvement in osteoblast functions and inhibition of osteoclast differentiation. Thus, alendronate-eluting chitosan substrates are promising materials for enhancing osteoblast functions and inhibiting osteoclast differentiation in orthopedic and dental fields.     

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing. Three-dimensional printed PLA/HA, β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1). Moreover, the biocompatibility, bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo. The defects were evaluated by micro-computed tomography and histological analysis at four and eight weeks after surgery, respectively. The results showed that each of the studied scaffolds had its own specific merits and drawbacks. Three-dimensional printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSC cell proliferation and differentiation to osteogenic cells. The outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference in inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery.     

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.     

Assessment of cell viability in a three-dimensional enzymatically cross-linked collagen scaffold

Microbial transglutaminase (mTGase) is an enzyme that introduces a covalent bond between peptide bound glutamine and lysine residues. Proteins cross-linked in this manner are often more resistant to proteolytic degradation and show increased tensile strength. This study evaluates the effects of mTGase mediated cross-linking of collagen on the cellular morphology, behaviour and viability of murine 3T3 fibroblasts following their seeding into collagen scaffolds. Additionally, cell mediated scaffold contraction, porosity and level of cross-linking of the scaffold has been analysed using image analysis software, scanning electron microscopy (SEM), colorimetric assays, and Fourier transform infrared spectroscopy (FTIR). We demonstrate that the biocompatibility and cellular morphology, when comparing cultures of fibroblasts integrated in mTGase cross-linked collagen scaffolds with the native collagen counterparts, remained unaffected. It has been also elicited that the structural characteristics of collagen have been preserved while introducing enzymatically resistant covalent bonds. This work was performed in the National Centre for Biomedical Engineering Science, Orbsen Building, National University of Ireland, Galway, Ireland.     

Hydroxyapatite (HA) bone scaffolds with controlled macrochannel pores

Hydroxyapatite (HA) macrochanneled porous scaffolds, with a controlled pore structure, were fabricated via a combination of the extrusion and lamination processes. The scaffold was architectured by aligning and laminating the extruded HA and carbon filaments. The macrochannel pores were formed by removing the carbon filaments after thermal treatments (binder removal and sintering). The porosity of the scaffolds was varied between 48 and 73% with a controlled pore size of ∼450 μm, by adjusting the fractions of HA and carbon filaments. As the porosity was increased from 48 to 73%, the compressive strength decreased from 11.5 to 3.2 MPa. However, the osteoblast-like cell responses on the scaffold, such as the proliferation rate and alkaline phosphatase (ALP) activity, were significantly enhanced as the porosity was increased.