Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate–gelatine–hydroxyapatite scaffolds with anisotropic pore structure

Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube‐like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube‐like pores were generated by diffusion‐controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 µm. To evaluate the potential use of alginate–gelatine–HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate–gelatine–HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10–14‐fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic‐induced hBMSCs. Furthermore, the expression of bone‐related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic‐induced hBMSCs on alginate–gelatin–HA scaffolds. Copyright © 2008 John Wiley & Sons, Ltd.     

Biomimetic scaffolds and dynamic compression enhance the properties of chondrocyte‐ and MSC‐based tissue‐engineered cartilage

Adult chondrocytes are surrounded by a protein‐ and glycosaminoglycan‐rich extracellular matrix and are subjected to dynamic mechanical compression during daily activities. The extracellular matrix and mechanical stimuli play an important role in chondrocyte biosynthesis and homeostasis. In this study, we aimed to develop scaffold and compressive loading conditions that mimic the native cartilage micro‐environment and enable enhanced chondrogenesis for tissue engineering applications. Towards this aim, we fabricated porous scaffolds based on silk fibroin (SF) and SF with gelatin/chondroitin sulfate/hyaluronate (SF‐GCH), seeded the scaffolds with either human bone marrow mesenchymal stromal cells (BM‐MSCs) or chondrocytes, and evaluated their performance with and without dynamic compression. Human chondrocytes derived from osteoarthritic joints and BM‐MSCs were seeded in scaffolds, precultured for 1 week, and subjected to compression with 10% dynamic strain at 1 Hz, 1 hr/day for 2 weeks. When dynamic compression was applied, chondrocytes significantly increased expression of aggrecan (ACAN) and collagen X (COL10A1) up to fivefold higher than free‐swelling controls. In addition, dynamic compression dramatically improved the chondrogenesis and chondrocyte biosynthesis cultured in both SF and SF‐GCH scaffolds evidenced by glycosaminoglycan (GAG) content, GAG/DNA ratio, and immunostaining of collagen type II and aggrecan. However, both chondrocytes and BM‐MSCs cultured in SF‐GCH scaffolds under dynamic compression showed higher GAG content and compressive modulus than those in SF scaffolds. In conclusion, the micro‐environment provided by SF‐GCH scaffolds and dynamic compression enhances chondrocyte biosynthesis and matrix accumulation, indicating their potential for cartilage tissue engineering applications.     

Fine‐tuning scaffolds for tissue regeneration: effects of formic acid processing on tissue reaction to silk fibroin

Formic acid (FA) plays a key role in the preparation of silk fibroin (SF) scaffolds from cocoons of Bombyx mori and is used for fibre distribution. In this study, we used a subcutaneous implantation model in Wistar rats to examine SF scaffolds prepared by treating the degummed cocoon with FA for either 30 or 60 min. The tissue reaction and inflammatory response to SF was assessed by qualitative histology at intervals from 3 to 180 days. Additionally, dynamic biomaterial‐induced vascularization and biomaterial degradation were quantified using a technique for analysing an image of the entire implanted biomaterial. Varying the FA treatment time led to different scaffold morphologies and resulted in two distinct peri‐implant tissue reactions. The 30 min‐treated scaffold was integrated into the surrounding tissue beginning at day 3 after implantation and vascularization increased 10‐fold from 15 to 180 days, while the scaffold was continuously degraded throughout the first 90 days. In contrast, the 60 min‐treated SF scaffold appeared as bulk for the first 90 days after implantation, after which a rapid degradation and vascularization process began. After 180 days, the tissue response was similar for both scaffolds, with eventual formation of a well vascularized connective tissue integrating the SF fibres. This study indicates that by modifying the FA treatment time, the tissue reaction to SF scaffolds can be tailored for different tissue‐engineering applications. The tunability and biocompatibility of SF make it an attractive scaffold for exploration in regenerative medicine and clinical tissue engineering. Copyright © 2010 John Wiley & Sons, Ltd.     

Role of electrospun fibre diameter and corresponding specific surface area (SSA) on cell attachment

In order to develop scaffolds for tissue regeneration applications, it is important to develop an understanding of the kinetics of cell attachment as a function of scaffold geometry. In the present study, we investigated how the specific surface area of electrospun scaffolds affected cell attachment and spreading. Number of cells attached to the scaffold was measured by the relative intensity of a metabolic dye (MTS) and cell spreading was analysed for individual cells by measuring the area of projected F‐actin cytoskeleton. We varied the fibre diameter to obtain a specific surface area distribution in the range 2.24–18.79 µ m^?1. In addition, we had one case where the scaffolds had beads in them and therefore had non‐uniform fibres. For each of these different geometries, we varied the cell‐seeding density (0.5–1 × 10⁵) and the serum concentration (0–12%) over the first 8 h in an electrospun polycaprolactone NIH 3T3 fibroblast system. Cells on beaded scaffolds showed the lowest attachment and almost no F‐actin spreading in all experiments indicating uniform fibre diameter is essential for electrospun scaffolds. For the uniform fibre scaffolds, cell attachment was a function of scaffold specific surface area (SSA) (18.79–2.24 µ m^?1) and followed two distinct trends: when scaffold SSA was < 7.13 µ m^?1, cell adhesion rate remained largely unchanged; however, for SSA > 7.13 µ m^?1 there was a significant increase in cellular attachment rate with increasing SSA. This indicated that nanofibrous scaffolds increased cellular adhesion compared to microfibrous scaffolds. This phenomenon is true for serum concentrations of 7.5% and higher. For 5% and lower serum concentration, cell attachment is low and higher SSA fails to make a significant improvement in cell attachment. When cell attachment was investigated at a single‐cell level by measuring the projected actin area, a similar trend was noted where the effect of higher SSA led to higher projected area for cells at 8 h. These results indicate that uniform electrospun scaffolds with SSA provide a faster cell attachment compared to lower SSA and beaded scaffolds. These results indicate that continuous electrospun nanofibrous scaffolds may be a good substrate for rapid tissue regeneration. Copyright © 2009 John Wiley & Sons, Ltd.     

Bone marrow stromal cells enhance the osteogenic properties of hydroxyapatite scaffolds by modulating the foreign body reaction

We aimed to investigate the osteogenic properties of bone marrow stromal cell (BMSC)‐loaded biomimetic constructs composed of hydroxyapatite (HA), with or without in vitro cell‐derived extracellular matrix (HA‐ECM), and to assess the cellular components of the elicited foreign body reaction. HA‐ECM constructs were produced by adult rat dermal fibroblasts cultured on top of synthetic HA microparticles. Rat calvarial critical‐sized defects (8 mm) were created and treated with the generated HA‐ECM constructs or HA microparticles, alone or combined with green fluorescent protein (GFP)‐expressing BMSCs. The new bone formation and the local cellular inflammatory response (macrophages, neutrophils, lymphocytes, eosinophils and PCNA‐index) were assessed by histomorphometry and immunohistochemistry at 2 and 12 weeks postoperatively. In addition, the BMSCs' survival and engraftment were checked. The largest volume of the newly formed bone was found in defects treated with HA‐ECM constructs combined with BMSCs (p < 0.05). Moreover, the implanted BMSCs modulated the local inflammatory response, demonstrated by either a significant increase (HA vs HA + BMSCs) or decrease (HA‐ECM vs HA‐ECM + BMSCs) of the inflammatory cell number. No donor BMSCs were detected at the site of implantation or in the host bone marrow at 2 or 12 weeks postoperatively. In conclusion, the treatment of critical‐sized calvarial defects with the BMSC‐loaded biomimetic constructs has significantly enhanced bone repair by modulating the foreign body reaction. Our findings highlight the implications of BMSCs in the regulation of the foreign body reaction triggered by tissue‐engineered constructs, proving a higher efficiency for the BMSC combination therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Generation of tunable glycosaminoglycan hydrogels to mimic extracellular matrices

Biomaterials and, especially, scaffolds may function as temporary extracellular matrix (ECM), mimicking in vivo environmental structures and facilitating cell growth and tissue regeneration. ECM is composed mostly of glycosaminoglycans (GAGs) and proteins, the ratio of GAGs, hyaluronic acid (HA):sulphated GAGs (sGAGs) being characteristic of each type of tissue. Umbilical cord (UC) and particularly Wharton's jelly (WJ) have been proposed as good sources for obtaining GAGs. In this work, we present a novel methodology for the extraction, purification and separation of GAGs from UC obtained from two different species, human and pig. The new methodology is based on enzymatic digestion of WJ, precipitation of GAGs with organic solvents, purification steps and chromatographic separation of GAGs using ion exchange columns. This novel process allows highly purified HA and sGAGs to be obtained from human and pig WJ. The composition of sGAGs and molecular weight of HA were very similar in the two species and GAGs are haemocompatible and non‐cytotoxic. Finally, these new biomaterials have significant bioactive properties, increasing proliferation rates of two cell lines, human adipose mesenchymal stem cells (ASCs) and fibroblasts. In summary, the separation of HA and sGAGs, linked to the improvement in the GAG quantification method described in this paper, opens new avenues for the formulation of natural biomaterials with various ratios of GAGs, mimicking tissue matrix for different tissue‐engineering applications. Copyright © 2014 John Wiley & Sons, Ltd.     

A composite material model for improved bone formation

The combination of synthetic polymers and calcium phosphates represent an improvement in the development of scaffolds for bone‐tissue regeneration. Ideally, these composites provide both mechanically and architecturally enhanced performances; however, they often lack properties such as osteoconductivity and cell bioactivation. In this study we attempted to generate a composite bone substitute maximizing the available osteoconductive surface for cell adhesion and activity. Highly porous scaffolds were prepared through a particulate leaching method, combining poly‐ε‐caprolactone (PCL) and hydroxyapatite (HA) particles, previously coated with a sucrose layer, to minimize their embedding by the polymer solution. Composite performances were evaluated both in vitro and in vivo. In PCL–sucrose‐coated HA samples, the HA particles were almost completely exposed and physically distinct from the polymer mesh, while uncoated control samples showed ceramic granules massively covered by the polymer. In vivo results revealed a significant extent of bone deposition around all sucrose‐coated HA granules, while only parts of the control uncoated HA granules were surrounded by bone matrix. These findings highlight the possibility of generating enhanced osteoconductive materials, basing the scaffold design on physiological and cellular concepts. Copyright © 2010 John Wiley & Sons, Ltd.     

Direct 3‐D printing of Ti‐6Al‐4V/HA composite porous scaffolds for customized mechanical properties and biological functions

Customized scaffold plays an important role in bone tissue regeneration. Precise control of the mechanical properties and biological functions of scaffolds still remains a challenge. In this study, metal and ceramic biomaterials are composited by direct 3‐D printing. Hydroxyapatite (HA) powders with diameter of about 25 μm and Ti‐6Al‐4V powders with diameter of 15–53 μm were mixed and modulated for preparing 3‐D printing inks formulation. Three different proportions of 8, 10, and 25 wt.% HA specimens were printed with same porosity of 72.1%. The green bodies of the printed porous scaffolds were sintered at 1,150°C in the atmosphere of argon furnace and conventional muffle furnace. The porosities of the final 3‐D‐printed specimens were 64.3 ± 0.8% after linear shrinkage of 6.5 ± 0.8%. The maximum compressive strength of the 3‐D‐printed scaffolds can be flexibly customized in a wide range. The maximum compressive strength of these scaffolds in this study ranged from 3.07 to 60.4 MPa, depending on their different preparation process. The phase composition analysis and microstructure characterization indicated that the Ti‐6Al‐4V and HA were uniformly composited in the scaffolds. The cytocompatibility and osteogenic properties were evaluated in vitro with rabbit bone marrow stromal cells (rBMSCs). Differentiation and proliferation of rBMSCs indicated good biocompatibility of the 3‐D‐printed scaffolds. The proposed 3‐D printing of Ti‐6Al‐4V/HA composite porous scaffolds with tunable mechanical and biological properties in this study is a promising candidate for bone tissue engineering.     

Behaviour of human mesenchymal stem cells on chemically synthesized HA–PCL scaffolds for hard tissue regeneration

Our goal was to characterize the response of human mesenchymal stem cells (hMSCs) to a novel composite scaffold for bone tissue engineering. The hydroxyapatite–polycaprolactone (HA–PCL) composite scaffolds were prepared by a sol–gel method at room temperature and the scaffold morphology was investigated by scanning electron microscopy (SEM)/energy‐dispersive spectroscopy (EDS) to validate the synthesis process. The response of two different lines of hMSCs, bone‐marrow‐derived human mesenchymal stem cells (BMSCs) and dental pulp stem cells (DPSCs) in terms of cell proliferation and differentiation into the osteoblastic phenotype, was evaluated using Alamar blue assay, SEM, histology and alkaline phosphatase activity. Our results indicate that tissue engineering by means of composite HA–PCL scaffolds may represent a new therapeutic strategy to repair craniofacial bone defects. Copyright © 2013 John Wiley & Sons, Ltd.     

Functionalized PVA–silk blended nanofibrous mats promote diabetic wound healing via regulation of extracellular matrix and tissue remodelling

Chronic cutaneous ulcers, a complex pathophysiological diabetic condition, represent a critical clinical challenge in the current diabetes mellitus pandemic. Consequently, there is a compelling need for bioactive dressings that can trigger healing processes for complete wound repair. Silk fibroin (SF), a natural protein polymer from mulberry and non‐mulberry silkworms, has properties that support accelerated wound healing rate. SF from non‐mulberry variety possesses additional cell‐binding motifs (arginine, glycine, and aspartate), offering cell–material interactions. This study is aimed to investigate wound healing efficacy of dressings made up of various SF varieties blended with poly(vinyl alcohol) biopolymer in alloxan‐induced diabetic rabbit model. The nanofibrous mats have been developed using electrospinning and functionalized with growth factors and LL‐37 antimicrobial peptide for sustained delivery. Following post 14‐day treatment, non‐mulberry SF (NMSF)‐based dressings healed the wounds faster, in comparison with their mulberry Bombyx mori SF, poly(vinyl alcohol), and control counterparts (p < .01). NMSF‐based dressings also supported faster granulation tissue development, angiogenesis, and reepithelialization of wounds. Gene expression study of matrix metalloproteinases and collagen proteins affirmed higher extent of tissue remodelling during the repair process. Furthermore, there was organized extracellular matrix deposition (collagen type I, collagen type III, elastin, and reticulin) and higher wound breaking strength in NMSF compared with other groups after 4 weeks. These results validated the potential of NMSF‐based bioactive dressings to regulate extracellular matrix deposition leading to faster and complete repair of chronic diabetic cutaneous wounds.     

Spatial distribution and survival of human and goat mesenchymal stromal cells on hydroxyapatite and β‐tricalcium phosphate

The combination of scaffolds and mesenchymal stromal cells (MSCs) is a promising approach in bone tissue engineering (BTE). Knowledge on the survival, outgrowth and bone‐forming capacity of MSCs in vivo is limited. Bioluminescence imaging (BLI), histomorphometry and immunohistochemistry were combined to study the fate of gene‐marked goat and human MSCs (gMSCs, hMSCs) on scaffolds with different osteoinductive properties. Luciferase–GFP‐labelled MSCs were seeded on hydroxyapatite (HA) or β‐tricalcium phosphate (TCP), cultured for 7 days in vitro in osteogenic medium, implanted subcutaneously in immunodeficient mice and monitored with BLI for 6 weeks. The constructs were retrieved and processed for histomorphometry and detection of luciferase‐positive cells (LPCs). For gMSCs, BLI revealed doubling of signal after 1 week, declining to 60% of input after 3 weeks and remaining constant until week 6. hMSCs showed a constant decrease of BLI signal to 25% of input, indicating no further expansion. Bone formation of gMSCs was two‐fold higher on TCP than HA. hMSCs and gMSCs control samples produced equal amounts of bone on TCP. Upon transduction, there was a four‐fold reduction in bone formation compared with untransduced hMSCs, and no bone was formed on HA. LPCs were detected at day 14, but were much less frequent at day 42. Striking differences were observed in spatial distribution. MSCs in TCP were found to be aligned and interconnected on the surface but were scattered in an unstructured fashion in HA. In conclusion, the spatial distribution of MSCs on the scaffold is critical for cell–scaffold‐based BTE. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of nanotubes and apatite on growth factor release from PLLA scaffolds

There is an evident clinical need for artificial bone restorative materials. In this respect, novel composites based on poly(L ‐lactic acid) (PLLA) have been described. The bone response of such polymer‐based composites is usually improved by the addition of bone morphogenetic protein‐2 (BMP‐2). However, released BMP‐2 is cleared almost immediately from the site of implantation by diffusion, whereas a prolonged retention of BMP‐2 onto the scaffold has been suggested to be more favourable. Besides the ability to improve the mechanical strength and osteoconductivity of polymeric scaffolds, both carbon nanotubes (CNTs) and microhydroxyapatite (µHA) have been described to facilitate such retention of BMP‐2 when incorporated into a composite scaffold. Therefore, in the current study, radiolabelled BMP‐2 was loaded onto plain PLLA and composite PLLA–CNT–µHA scaffolds. Subsequently, the scaffolds were implanted subcutaneously for 5 weeks in rats and BMP‐2 release was measured. Release started with an initial phase of quick release, followed by a gradual release of BMP‐2. Both scaffold types comprised the same in vivo release properties for BMP‐2. The bioactivity of the BMP‐2 remained unaltered. It can be concluded that incorporated CNTs and µHA did not affect BMP‐2 release from composite scaffold materials. Copyright © 2010 John Wiley & Sons, Ltd.     

PCL–HA microscaffolds for in vitro modular bone tissue engineering

The evolution of microscaffolds and bone‐bioactive surfaces is a pivotal point in modular bone tissue engineering. In this study, the design and fabrication of porous polycaprolactone (PCL) microscaffolds functionalized with hydroxyapatite (HA) nanoparticles by means of a bio‐safe and versatile thermally‐induced phase separation process is reported. The ability of the as‐prepared nanocomposite microscaffolds to support the adhesion, growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in standard and osteogenic media and using dynamic seeding/culture conditions was investigated. The obtained results demonstrated that the PCL–HA nanocomposite microparticles had an enhanced interaction with hMSCs and induced their osteogenic differentiation, even without the exogenous addition of osteogenic factors. In particular, calcium deposition, alizarin red assay, histological analysis, osteogenic gene expression and collagen I secretion were assessed. The results of these tests demonstrated the formation of bone microtissue precursors after 28 days of dynamic culture. These findings suggest that PCL–HA nanocomposite microparticles represent an excellent platform for in vitro modular bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.     

Artificial extracellular matrices support cell growth and matrix synthesis of human dermal fibroblasts in macroporous 3D scaffolds

Surface modification of materials designed for regenerative medicine may improve biocompatibility and functionality. The application of glycosaminoglycans (GAGs) and chemically sulphated GAG derivatives is a promising approach for designing functional biomaterials, since GAGs interact with cell‐derived growth factors and have been shown to support fibroblast growth in two‐dimensional (2D) cultures. Here, coatings with artificial extracellular matrix (aECM), consisting of the structural protein collagen I and the GAG hyaluronan (HA) or sulphated HA derivatives, were investigated for their applicability in a three‐dimensional (3D) system. As a model, macroporous poly(lactic‐co‐glycolic acid) (PLGA) scaffolds were homogeneously coated with aECM. The resulting scaffolds were characterized by compressive moduli of 0.9–1.2 MPa and pore sizes of 40–420 µm. Human dermal fibroblasts (dFbs) colonized these aECM‐coated PLGA scaffolds to a depth of 400 µm within 14 days. In aECM‐coated scaffolds, collagen I(α1) and collagen III(α1) mRNA expression was reduced, while matrix metalloproteinase‐1 (MMP‐1) mRNA expression was increased within 7 days, suggesting matrix‐degradation processes. Stimulation with TGFβ1 generally increased cell density and collagen synthesis, demonstrating the efficiency of bioactive molecules in this 3D model. Thus, aECM with sulphated HA may modulate the effectivity of TGFβ1‐induced collagen I(α1) expression, as demonstrated previously in 2D systems. Overall, the tested aECM with modified HA is also a suitable material for fibroblast growth under 3D conditions. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhanced osteogenesis of human mesenchymal stem cells by self‐assembled peptide hydrogel functionalized with glutamic acid templated peptides

Self‐assembling peptide (SAP) hydrogel has been shown to be an excellent biological material for three‐dimensional cell culture and stimulatie cell migration and differentiation into the scaffold, as well as for repairing bone tissue defects. Herein, we designed one of the SAP scaffolds KLD (KLDLKLDLKLDL) through direct coupling to short bioactive motif O1 (EEGGC) and O2 (EEEEE) of which bioactivity on osteogenic differentiation was previously demonstrated and self‐assembled in different concentrations (0.5%, 1%, and 2%). Our aim was to enhance osteogenesis and biomineralization of injectable SAP hydrogels with controlled mechanical properties so that the peptide hydrogel also becomes capable of being injected to bone defects. The molecular integration of the nanofibrous peptide scaffolds was observed using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The rheological properties and degradation profile of SAP hydrogels were evaluated to ensure stability of SAPs. Compared with pure KLD scaffold, we found that these designed bioactive peptide scaffolds significantly promoted hMSCs proliferation depicted by biochemical analysis of alkaline phosphatase (ALP) activity, total calcium deposition. Moreover, key osteogenic markers of ALP activity, collagen type I (COL‐1), osteopontin (OP), and osteocalcin (OCN) expression levels determined by real‐time polymerase chain reaction (PCR) and immunofluorescence analysis were also significantly increased with the addition of glutamic acid residues to KLD. We demonstrated that the designed SAP scaffolds promoted the proliferation and osteogenic differentiation of hMSCs. Our results suggest that these designed bioactive peptide scaffolds may be useful for promoting bone tissue regeneration.     

Additive manufacturing of poly[(R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate] scaffolds for engineered bone development

A wide range of poly(hydroxyalkanoate)s (PHAs), a class of biodegradable polyesters produced by various bacteria grown under unbalanced conditions, have been proposed for the fabrication of tissue‐engineering scaffolds. In this study, the manufacture of poly[(R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate] (or PHBHHx) scaffolds, by means of an additive manufacturing technique based on a computer‐controlled wet‐spinning system, was investigated. By optimizing the processing parameters, three‐dimensional scaffolds with different internal architectures were fabricated, based on a layer‐by‐layer approach. The resulting scaffolds were characterized by scanning electron microscopy, which showed good control over the fibre alignment and a fully interconnected porous network, with porosity in the range 79–88%, fibre diameter 47–76 µm and pore size 123–789 µm. Moreover, the resulting fibres presented an internal porosity connected to the external fibre surface as a consequence of the phase‐inversion process governing the solidification of the polymer solution. Scaffold compressive modulus and yield stress and strain could be varied in a certain range by changing the architectural parameters. Cell‐culture experiments employing the MC3T3‐E1 murine pre‐osteoblast cell line showed good cell proliferation after 21 days of culture. The PHBHHx scaffolds demonstrated promising results in terms of cell differentiation towards an osteoblast phenotype. Copyright © 2014 John Wiley & Sons, Ltd.     

Controlled release of vascular endothelial growth factor from spray‐dried alginate microparticles in collagen–hydroxyapatite scaffolds for promoting vascularization and bone repair

A major limitation with current tissue‐engineering approaches is creating functionally vascularized constructs that can successfully integrate with the host; this often leads to implant failure, due to avascular necrosis. In order to overcome this, the objective of the present work was to develop a method to incorporate growth factor‐eluting alginate microparticles (MPs) into freeze‐dried, collagen‐based scaffolds. A collagen–hydroxyapatite (CHA) scaffold, previously optimized for bone regeneration, was functionalized for the sustained delivery of an angiogenic growth factor, vascular endothelial growth factor (VEGF), with the aim of facilitating angiogenesis and enhancing bone regeneration. VEGF was initially encapsulated in alginate MPs by spray‐drying, producing particles of < 10 µm in diameter. This process was found to effectively encapsulate and control VEGF release while maintaining its stability and bioactivity post‐processing. These VEGF‐MPs were then incorporated into CHA scaffolds, leading to homogeneous distribution throughout the interconnected scaffold pore structure. The scaffolds were capable of sustained release of bioactive VEGF for up to 35 days, which was proficient at increasing tubule formation by endothelial cells in vitro. When implanted in vivo in a rat calvarial defect model, this scaffold enhanced vessel formation, resulting in increased bone regeneration compared to empty‐defect and VEGF‐free scaffolds. This biologically functionalized scaffold, composed entirely of natural‐based materials, may offer an ideal platform to promote angiogenesis and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

MgCHA particles dispersion in porous PCL scaffolds: in vitro mineralization and in vivo bone formation

In this work, we focus on the in vitro and in vivo response of composite scaffolds obtained by incorporating Mg,CO₃‐doped hydroxyapatite (HA) particles in poly(ε‐caprolactone) (PCL) porous matrices. After a complete analysis of chemical and physical properties of synthesized particles (i.e. SEM/EDS, DSC, XRD and FTIR), we demonstrate that the Mg,CO₃ doping influences the surface wettability with implications upon cell–material interaction and new bone formation mechanisms. In particular, ion substitution in apatite crystals positively influences the early in vitro cellular response of human mesenchymal stem cells (hMSCs), i.e. adhesion and proliferation, and promotes an extensive mineralization of the scaffold in osteogenic medium, thus conforming to a more faithful reproduction of the native bone environment than undoped HA particles, used as control in PCL matrices. Furthermore, we demonstrate that Mg,CO₃‐doped HA in PCL scaffolds support the in vivo cellular response by inducing neo‐bone formation as early as 2 months post‐implantation, and abundant mature bone tissue at the sixth month, with a lamellar structure and completely formed bone marrow. Together, these results indicate that Mg²⁺ and CO₃^2– ion substitution in HA particles enhances the scaffold properties, providing the right chemical signals to combine with morphological requirements (i.e. pore size, shape and interconnectivity) to drive osteogenic response in scaffold‐aided bone regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

Nanostructured gellan and xanthan hydrogel depot integrated within a baghdadite scaffold augments bone regeneration

Controlled delivery of biological cues through synthetic scaffolds to enhance the healing capacity of bone defects is yet to be realized clinically. The purpose of this study was development of a bioactive tissue‐engineered scaffold providing the sustained delivery of an osteoinductive drug, dexamethasone disodium phosphate (DXP), encapsulated within chitosan nanoparticles (CN). Porous baghdadite (BD; Ca₃ZrSi₂O₉) scaffolds, a zirconia‐modified calcium silicate ceramic, was coated with DXP‐encapsulated CN nanoparticles (DXP–CN) using nanostructured gellan and xanthan hydrogel (GX). Crosslinker and GX polymer concentrations were optimized to achieve a homogeneous distribution of hydrogel coating within BD scaffolds. Dynamic laser scattering indicated an average size of 521 ± 21 nm for the DXP–CN nanoparticles. In vitro drug‐release studies demonstrated that the developed DXP–CN–GX hydrogel‐coated BD scaffolds (DXP–CN–GX–BD) resulted in a sustained delivery of DXP over the 5 days (78 ± 6% of drug release) compared with burst release over 1 h, seen from free DXP loaded in uncoated BD scaffolds (92 ± 8% release in 1 h). To estimate the influence of controlled delivery of DXP from the developed scaffolds, the effect on MG 63 cells was evaluated using various bone differentiation assays. Cell culture within DXP–CN–GX–BD scaffolds demonstrated a significant increase in the expression of early and late osteogenic markers of alkaline phosphatase activity, collagen type 1 and osteocalcin, compared to the uncoated BD scaffold. The results suggest that the DXP‐releasing nanostructured hydrogel integrated within the BD scaffold caused sustained release of DXP, improving the potential for osteogenic differentiation. Copyright © 2015 John Wiley & Sons, Ltd.