In Vitro Bone Formation Associated with Apatite Coated Polylactide

Bone formation onto poly(L-lactide), which was plasma-spray coated with various quantities of hydroxyapatite (0%, 15%, 36% and 100% coverage), was investigated in an in vitro assay. Rat bone marrow cells were grown on the different coatings and the cellular response and elaborated extracellular matrix was examined at the light and electron microscopical level after 1, 2 , 4 and 8 weeks of culture. Proliferation of cells into multilayers was seen on the 0% , 36% and 100% , but not on the 15 % coatings. Coinciding with this was the sparse formation of extracellular matrix on the latter, and its abundant appearance on the former three coatings. Scanning and transmission electron microscopy revealed a mineralized extracellular matrix on the 100% and 36% coatings after 2 and 4 weeks , respectively, and on the 15 % coating after 8 weeks. Mineralization was not observed on uncoated poly(L-lactide). At the interface between hydroxyapatite and the mineralized extracellular matrix, one or more electron dense layers were frequently observed , which showed morphological similarities with structures between these two entities in vivo. The results of this in vitro study show that, in the model used, hydroxyapatite is required to obtain the elaboration of mineralized extracellular matrix on poly(L-lactide)

The Effect of PEO Ratio on Degradation, Calcification and Bone Bonding of PEO/PBT Copolymer (Polyactive)

In this study, we evaluated the effect of PEO/ PBT proportion on the behavior of a range of PEO/ PBT segmented copolymers (Polyactive) during subcutaneous and intrabony implantation in the rat. It was demonstrated that varying the PEO proportion affected degradation, calcification and bone-bonding. The PEO/PBT 70/30 and 60/40 showed extensive degradation after 1 year, PEO/PBT 55145 an intermediate degradation, and the 40/60 and 30170 copolymers showed little and hardly any degradation respectively. PEO content also affected the degree of calcification . PEO/PBT 70/30 showed extensive and early calcification whereas almost no calcification was seen with PEO/PBT 30170. Since calcified sites at the periphery of the polymeric implants were locations of preference for bone-bonding to occur, PEO/PBT proportion also influenced bone/PEO/PBT interactions. The materials with the highest PEO content most frequently showed morphological indications of bone-bonding , while the material with 30 % PEO showed no bone/biomaterial contact. The differences in bone-bonding activity were also reflected by the occurrence of an electron. dense zone at the bone-biomaterial interface which was morphologically similar to that observed for calcium phosphate ceramics

Influence of PCL molecular weight on mesenchymal stromal cell differentiation

Regenerating or replacing bone, chondral and osteochondral defects, is an active field in tissue engineering. A general strategy is to use a temporary scaffold in which cells are seeded onto the scaffold prior to implantation or attracted into the scaffold from surrounding tissues in the implantation site to form the desired tissue. Several biomaterials have been used for the fabrication of scaffolds, including polycaprolactone (PCL) which is often used for musculoskeletal tissue engineering. The effect of the PCL scaffold architecture on the cell behavior has been investigated. However, the mechanical properties of the bulk material were not taken into account in these studies. PCL is available in a range of molecular weights, resulting in a range of bulk mechanical properties. Since bulk material stiffness is able to direct cell differentiation, it is likely that the molecular weight of PCL may influence cell behavior. Here, we investigated the bulk material properties of both low and high molecular weight PCL scaffolds fabricated through additive manufacturing. The low molecular weight PCL showed a lower bulk material stiffness. During in vitro cell culture, this resulted in a stronger tendency for hypertrophic chondrogenic differentiation compared to the high molecular weight PCL. This study shows that apart from the polymer chemistry and scaffold architecture, the bulk mechanical properties of the polymer used is an important parameter in scaffold fabrication. This is an important finding for the optimization of osteochondral tissue engineering

Influence of Crystal Structure on the Establishment of the Bone-Calcium Phosphate Interface In Vitro

An in vitro rat bone marrow cell system was used to examine the interfacial ultrastructure established between various calcium phosphates and mineralized tissue. The investigated calcium phosphates comprised hydroxyapatite (HA), fluorapatite (FA), tricalcium phosphate (TCP), tetracalcium phosphate (TECP) and magnesium whitlockite (MWL). Both scanning and transmission electron microscopy were used to examine the elaborated interface. The time in which a mineralized extracellular matrix was formed on the various materials differed from 2 weeks on HA, TCP and TECP, to 8 weeks on FA. It was only occasionally observed in some areas on MWL, which might have been due to aluminum impurities in the coating. With transmission electron microscopy, three distinct interfacial structures were observed. They differed in the presence or absence of a collagen free, 0. 7 to 0. 8 J.Lm wide, amorphous zone and a 20 to 60 nm thick electron dense layer , interposed between the material surface and the mineralized extracellular matrix. The electron dense layer was considered to be at least partially caused by protein adsorption , which would precede or concur with biological mineralization events , while the amorphous zone was regarded to represent partial degradation of the calcium phosphate surfaces. The results of this study show that plasma sprayed calcium phosphates will display different bone-bonding and biodegradation properties , depending on their chemical composition and crystal structures

Triphasic scaffolds for the regeneration of the bone-ligament interface

A triphasic scaffold (TPS) for the regeneration of the bone-ligament interface was fabricated combining a 3D fiber deposited polycaprolactone structure and a polylactic co-glycolic acid electrospun. The scaffold presented a gradient of physical and mechanical properties which elicited different biological responses from human mesenchymal stem cells. Biological test were performed on the whole TPS and on scaffolds comprised of each single part of the TPS, considered as the controls. The TPS showed an increase of the metabolic activity with culturing time that seemed to be an average of the controls at each time point. The importance of differentiation media for bone and ligament regeneration was further investigated. Metabolic activity analysis on the different areas of the TPS showed a similar trend after 7 days in both differentiation media. Total alkaline phosphatase (ALP) activity analysis showed a statistically higher activity of the TPS in mineralization medium compared to the controls. A different glycosaminoglycans amount between the TPS and its controls was detected, displaying a similar trend with respect to ALP activity. Results clearly indicated that the integration of electrospinning and additive manufacturing represents a promising approach for the fabrication of scaffolds for the regeneration of tissue interfaces, such as the bone-to-ligament one, because it allows mimicking the structural environment combining different biomaterials at different scales

Hydrogels that listen to cells:a review of cell-responsive strategies in biomaterial design for tissue regeneration

The past decade has seen a decided move from static and passive biomaterials to biodegradable, dynamic, and stimuli responsive materials in the laboratory and the clinic. Recent advances towards the rational design of synthetic cell-responsive hydrogels-biomaterials that respond locally to cells or tissues without the input of an artificial stimulus-have provided new strategies and insights on the use of artificial environments for tissue engineering and regenerative medicine. These materials can often approximate responsive functions of a cell's complex natural extracellular environment, and must respond to the small and specific stimuli provided within the vicinity of a cell or tissue. In the current literature, there are three main cell-based stimuli that can be harnessed to create responsive hydrogels: (1) enzymes (2) mechanical force and (3) metabolites/small molecules. Degradable bonds, dynamic covalent bonds, and non-covalent or supramolecular interactions are used to provide responsive architectures that enable features ranging from cell selective infiltration to control of stem-cell differentiation. The growing ability to spatiotemporally control the behavior of cells and tissue with rationally designed responsive materials has the ability to allow control and autonomy to future generations of materials for tissue regeneration, in addition to providing understanding and mimicry of the dynamic and complex cellular niche

Stimulatory effects of inorganic ions on osteogenesis in vitro

Introduction: Several studies demonstrated the effect of silicate ions (Si) on differentiation of bone precursor cells1,2, although its exact role in processes related to bone formation and remodeling is still incompletely understood. The focus of this work is to explore the effect of calcium and silicate ions on growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs). This strategy may reduce the need for growth factors required to stimulate bone formation in regenerative approaches, decreasing the associated costs and overcoming stability issues. Materials and Methods In order to define the range of Si concentrations that are not toxic to cells, we performed a preliminary study varying Si concentrations from 0.00357mM to 4mM. The concentration of the Ca ions was selected based on the earlier study by Barradas et. al3. Cell culture media were supplemented by using sodium silicate (Na2SiO3) and/or calcium chloride dehydrate (CaCl2*2H2O) as Si and Ca precursors, respectively. hMSCs derived from bone marrow were seeded at a seeding density of 2.000 cells/cm2 and allowed to adhere overnight. Then, the medium was replaced by the appropriate supplemented medium and cells were cultured for 3, 7, 14 and 18 days. Basic and osteogenic media were used as negative and positive controls. Cell proliferation was evaluated by DNA quantification. hMSCs osteogenic gene expression was evaluated by Q-PCR. Results DNA quantification indicated an increase in cell number during the culture time for all the conditions. Results obtained by Q-PCR revealed a significantly higher expression of osteocalcin (OC) and bone morphogenetic protein-2 (BMP2) in cells cultured in media supplemented by both ions, as compared to media containing either Ca or Si alone. Discussion and Conclusions DNA quantification studies indicated that none of the selected concentrations had a negative influence on cell proliferation. The increase in osteogenic gene expression for cells cultured with both Ca and Si suggested a synergistic effect of the two ions on osteogenic differentiation of hMSCs. We further showed that cells cultured in the medium with the highest concentration of Ca (7.8mM) revealed a higher expression of the selected genes, which is in accordance with the earlier results by Barradas et al3. The obtained results suggest the importance of combining both ions, Ca and Si, for promoting the osteogenic differentiation of hMSCs. References 1. Hoppe A, Biomaterials 32: 2757-2774, 2011. 2. Beck Jr GR, Nanomedicine: Nanotechnology, Biology, and Medicine,1-11, 2011 3. Barradas AMC et al., Biomaterials 3205-3215, 2012. Acknowledgments The author thanks the Portuguese Foundation for Science and Technology (FCT) for the grant (SFRH/BD/69962/2010). Disclosures The authors have nothing to disclose

Cytocompatibility and response of osteoblastic-like cells to starch-based polymers : effect of several additives and processing conditions

This work reports on the biocompatibility evaluation of new biodegradable starch-based polymers that are under consideration for use in orthopaedic temporary applications and as tissue engineering scaffolds. It has been shown in previous works that by using these polymers it is both possible to produce polymer/hydroxyapatite (HA) composites (with or without the use of coupling agents) with mechanical properties matching those of the human bone, and to obtain 3D structures generated by solid blowing agents, that are suitable for tissue engineering applications. This study was focused on establishing the influence of several additives (ceramic fillers, blowing agents and coupling agents) and processing methods/conditions on the biocompatibility of the materials described above. The cytotoxicity of the materials was evaluated using cell culture methods, according to ISO/EN 109935 guidelines. A cell suspension of human osteosarcoma cells (HOS) was also seeded on a blend of corn starch with ethylene vinyl alcohol (SEVA-C) and on SEVA-C/HA composites, in order to have a preliminary indication on cell adhesion and proliferation on the materials surface. In general, the obtained results show that all the different materials based on SEVA-C, (which are being investigated for use in several biomedical applications), as well as all the additives (including the novel coupling agents) and different processing methods required to obtain the different properties/products, can be used without inducing a cytotoxic behaviour to the developed biomaterial

A Comparative Study of the Interactions of Two Calcium Phosphates, PEO/PBT Copolymer (Polyactive) and a Silicone Rubber with Bone and Fibrous Tissue

In this study, hydroxyapatite, tetracalcium phosphate, HPEO/PBT 55145 copolymer, PEO/PBT 55!45 copolymer (Polyactive) and silicone rubber were implanted as dense blocks, subcutaneously and. into the tibia of rats. Biocompatibility and degradation were investigated but most attention was directed to .the bone/biomaterial interactions. None of the materials showed any significant adverse tissue reactions. With exception of the silicone rubber, all materials sho~ed bone bonding phenomena based on both morphological and mechanical evaluations. (H)PEO/PBT 55145 copolymer is the first polymer reported to be bonded by bone and thus widens the spectrum of bone bonding materials with a low modulus, degradable, elastomer in contrast to the high modulus glasses and ceramics that are available to date. The possible associated bone-bonding mechanism is briefly discussed