Roman A. Perez

Naturally and synthetic smart composite biomaterials for tissue regeneration

The development of smart biomaterials for tissue regeneration has become the focus of intense research interest. More opportunities are available by the composite approach of combining the biomaterials in the form of biopolymers and/or bioceramics either synthetic or natural. Strategies to provide smart capabilities to the composite biomaterials primarily seek to achieve matrices that are instructive/inductive to cells, or that stimulate/trigger target cell responses that are crucial in the tissue regeneration processes. Here, we review in-depth, recent developments concerning smart composite biomaterials available for delivery systems of biofactors and cells and scaffolding matrices in tissue engineering. Smart composite designs are possible by modulating the bulk and surface properties that mimic the native tissues, either in chemical (extracellular matrix molecules) or in physical properties (e.g. stiffness), or by introducing external therapeutic molecules (drugs, proteins and genes) within the structure in a way that allows sustainable and controllable delivery, even time-dependent and sequential delivery of multiple biofactors. Responsiveness to internal or external stimuli, including pH, temperature, ionic strength, and magnetism, is another promising means to improve the multifunctionality in smart scaffolds with on-demand delivery potential. These approaches will provide the next-generation platforms for designing three-dimensional matrices and delivery systems for tissue regenerative applications.

New processing approaches in calcium phosphate cements and their applications in regenerative medicine

The key feature of calcium phosphate cements (CPCs) lies in the setting reaction triggered by mixing one or more solid calcium phosphate salts with an aqueous solution. Upon mixture, the reaction takes place through a dissolution-precipitation process which is macroscopically observed by a gradual hardening of the cement paste. The precipitation of hydroxyapatite nanocrystals at body or room temperature, and the fact that those materials can be used as self-setting pastes, have for many years been the most attractive features of CPCs. However, the need to develop materials able to sustain bone tissue ingrowth and be capable of delivering drugs and bioactive molecules, together with the continuous requirement from surgeons to develop more easily handling cements, has pushed the development of new processing routes that can accommodate all these requirements, taking advantage of the possibility of manipulating the self-setting CPC paste. It is the goal of this paper to provide a brief overview of the new processing developments in the area of CPCs and to identify the most significant achievements.

Intrinsic porosity of calcium phosphate cements and its significance for drug delivery and tissue engineering applications.

One key point in the field of tissue engineering and drug delivery is to provide materials with an adequate porosity. Many events, including nutrient and waste exchange in scaffolds for tissue engineering, as well as the drug-loading capacity and control of the release rate in drug delivery systems, are controlled by the size, shape and distribution of the pores in the material. Calcium phosphate cements (CPCs) possess an intrinsic porosity that is highly suited for these applications, and this porosity can be controlled by modifying some processing parameters. The objective of this work was to characterize and control the intrinsic porosity of alpha-tricalcium phosphate (alpha-TCP) cements, and to investigate its role against adsorption of bovine serum albumin (BSA). Cements with different percentages of open porosity (35-55%) were prepared by modifying the liquid-to-powder ratio. In addition, two different TCP particles were used to yield cements with specific surface areas of approximately 20 and approximately 37m(2)g(-1). Mercury porosimetry analysis on the set cements showed in most cases a bimodal pore size distribution which varied with the processing parameters and affected differently the adsorption and penetration of BSA. The peak occurring at larger pore dimensions controlled the penetration of BSA and was ascribed to the voids generated in between crystal aggregates, while the peak appearing at lower pore sizes was believed to be due to the intercrystallite voids within aggregates. It was found that, at the concentrations studied, the high intrinsic porosity in CPC does not ensure protein penetration unless there is an adequate pore size distribution.

Silica-based mesoporous nanoparticles for controlled drug delivery

Drug molecules with lack of specificity and solubility lead patients to take high doses of the drug to achieve sufficient therapeutic effects. This is a leading cause of adverse drug reactions, particularly for drugs with narrow therapeutic window or cytotoxic chemotherapeutics. To address these problems, there are various functional biocompatible drug carriers available in the market, which can deliver therapeutic agents to the target site in a controlled manner. Among the carriers developed thus far, mesoporous materials emerged as a promising candidate that can deliver a variety of drug molecules in a controllable and sustainable manner. In particular, mesoporous silica nanoparticles are widely used as a delivery reagent because silica possesses favourable chemical properties, thermal stability and biocompatibility. Currently, sol-gel-derived mesoporous silica nanoparticles in soft conditions are of main interest due to simplicity in production and modification and the capacity to maintain function of bioactive agents. The unique mesoporous structure of silica facilitates effective loading of drugs and their subsequent controlled release. The properties of mesopores, including pore size and porosity as well as the surface properties, can be altered depending on additives used to fabricate mesoporous silica nanoparticles. Active surface enables functionalisation to modify surface properties and link therapeutic molecules. The tuneable mesopore structure and modifiable surface of mesoporous silica nanoparticle allow incorporation of various classes of drug molecules and controlled delivery to the target sites. This review aims to present the state of knowledge of currently available drug delivery system and identify properties of an ideal drug carrier for specific application, focusing on mesoporous silica nanoparticles.

Polymeric additives to enhance the functional properties of calcium phosphate cements

The vast majority of materials used in bone tissue engineering and regenerative medicine are based on calcium phosphates due to their similarity with the mineral phase of natural bone. Among them, calcium phosphate cements, which are composed of a powder and a liquid that are mixed to obtain a moldable paste, are widely used. These calcium phosphate cement pastes can be injected using minimally invasive surgery and adapt to the shape of the defect, resulting in an entangled network of calcium phosphate crystals. Adding an organic phase to the calcium phosphate cement formulation is a very powerful strategy to enhance some of the properties of these materials. Adding some water-soluble biocompatible polymers in the calcium phosphate cement liquid or powder phase improves physicochemical and mechanical properties, such as injectability, cohesion, and toughness. Moreover, adding specific polymers can enhance the biological response and the resorption rate of the material. The goal of this study is to overview the most relevant advances in this field, focusing on the different types of polymers that have been used to enhance specific calcium phosphate cement properties.

Core–shell designed scaffolds for drug delivery and tissue engineering

Scaffolds that secure and deliver therapeutic ingredients like signaling molecules and stem cells hold great promise for drug delivery and tissue engineering. Employing a core-shell design for scaffolds provides a promising solution. Some unique methods, such as co-concentric nozzle extrusion, microfluidics generation, and chemical confinement reactions, have been successful in producing core-shelled nano/microfibers and nano/microspheres. Signaling molecules and drugs, spatially allocated to the core and/or shell part, can be delivered in a controllable and sequential manner for optimal therapeutic effects. Stem cells can be loaded within the core part on-demand, safely protected from the environments, which ultimately affords ex vivo culture and in vivo tissue engineering. The encapsulated cells experience three-dimensional tissue-mimic microenvironments in which therapeutic molecules are secreted to the surrounding tissues through the semi-permeable shell. Tuning the material properties of the core and shell, changing the geometrical parameters, and shaping them into proper forms significantly influence the release behaviors of biomolecules and the fate of the cells. This topical issue highlights the immense usefulness of core-shell designs for the therapeutic actions of scaffolds in the delivery of signaling molecules and stem cells for tissue regeneration and disease treatment.

Role of pore size and morphology in musculo-skeletal tissue regeneration

Biomaterials in the form of scaffolds hold great promise in the regeneration of diseased tissues. The scaffolds stimulate cellular adhesion, proliferation and differentiation. While the scaffold composition will dictate their biocompatibility, their porosity plays a key role in allowing proper cell penetration, nutrient diffusion as well as bone ingrowth. Porous scaffolds are processed with the help of a wide variety of techniques. Designing scaffolds with the appropriate porosity is a complex issue since this may jeopardize other physico-chemical properties. From a macroscopic point of view, parameters such as the overall architecture, pore morphology, interconnectivity and pore size distribution, have unique roles in allowing bone ingrowth to take place. From a microscopic perspective, the adsorption and retention of proteins in the microporosities of the material will dictate the subsequent cell adhesion. Therefore, the microstructure of the substrate can determine cell proliferation as well as the expression of specific osteogenic genes. This review aims at discussing the effect of micro- and macroporosity on the physico-chemical and biological properties of scaffolds for musculo-skeletal tissue regeneration.

Porous hydroxyapatite and gelatin/hydroxyapatite microspheres obtained by calcium phosphate cement emulsion

Hydroxyapatite and hybrid gelatine/hydroxyapatite microspheres were obtained through a water in oil emulsion of a calcium phosphate cement (CPC). The setting reaction of the CPC, in this case the hydrolysis of α-tricalcium phosphate, was responsible for the consolidation of the microspheres. After the setting reaction, the microspheres consisted of an entangled network of hydroxyapatite crystals, with a high porosity and pore sizes ranging between 0.5 and 5 μm. The size of the microspheres was tailored by controlling the viscosity of the hydrophobic phase, the rotation speed, and the initial powder size of the CPC. The incorporation of gelatin increased the sphericity of the microspheres, as well as their size and size dispersion. To assess the feasibility of using the microspheres as cell microcarriers, Saos-2 cells were cultured on the microspheres. Fluorescent staining, SEM studies, and LDH quantification showed that the microspheres were able to sustain cell growth. Cell adhesion and proliferation was significantly improved in the hybrid gelatin/hydroxyapatite microspheres as compared to the hydroxyapatite ones.

Therapeutic bioactive microcarriers: co-delivery of growth factors and stem cells for bone tissue engineering.

Novel microcarriers made of sol-gel-derived bioactive glasses were developed for delivering therapeutic molecules effectively while cultivating stem cells for bone tissue engineering. Silica sols with varying concentration of Ca (0-30 mol.%) were formulated into microspheres ranging from 200 to 300 μm under optimized conditions. A highly mesoporous structure was created, with mesopore sizes of 2.5-6.3 nm and specific surface areas of 420-710 m(2)g(-1), which was highly dependent on the Ca concentration. Therapeutic molecules could be effectively loaded within the mesoporous microcarriers during microsphere formulation. Cytochrome C (cyt C), used as a model protein for the release study, was released in a highly sustainable manner, with an almost zero-order kinetics over a period of months; the amount released was ~2% at 9 days, and 15% at 40 days. A slight increase in the release rate was observed in the microcarrier containing Ca, which was related to the dissolution rate and pore size. The presence of Ca accelerated the formation of hydroxyapatite on the surface of the microcarriers. Cells cultured on the bioactive microcarriers were well adhered and distributed, and proliferated actively, confirming the three-dimensional substrate role of the microcarriers. An in vivo study performed in a rat subcutaneous model demonstrated the satisfactory biocompatibility of the prepared microspheres. As a therapeutic target molecule, basic fibroblast growth factor (bFGF) was incorporated into the microcarriers. A slow release pattern similar to that of cyt C was observed for bFGF. Cells adhered and proliferated to significantly higher levels on the bFGF-loaded microcarriers, demonstrating the effective role of bFGF in cell proliferative potential. It is believed that the developed mesoporous bioactive glass microspheres represent a new class of therapeutic cell delivery carrier, potentially useful in the sustainable delivery of therapeutic molecules such as growth factors, as well as in the support of stem cell proliferation and osteogenesis for bone tissue engineering.

Novel therapeutic core–shell hydrogel scaffolds with sequential delivery of cobalt and bone morphogenetic protein-2 for synergistic bone regeneration

UNLABELLED Enabling early angiogenesis is a crucial issue in the success of bone tissue engineering. Designing scaffolds with therapeutic potential to stimulate angiogenesis as well as osteogenesis is thus considered a promising strategy. Here, we propose a novel scaffold designed to deliver angiogenic and osteogenic factors in a sequential manner to synergize the bone regeneration event. Hydrogel fibrous scaffolds comprised of a collagen-based core and an alginate-based shell were constructed. Bone morphogenetic protein 2 (BMP2) was loaded in the core, while the shell incorporated Co ions, enabled by the alginate crosslinking in CoCl2/CaCl2 solution. The incorporation of Co ions was tunable by altering the concentration of Co ions in the crosslinking solution. The incorporated Co ions, that are known to play a role in angiogenesis, were released rapidly within a week, while the BMP2, acting as an osteogenic factor, was released in a highly sustainable manner over several weeks to months. The release of Co ions significantly up-regulated the in vitro angiogenic properties of cells, including the expression of angiogenic genes (CD31, VEGF, and HIF-1α), secretion of VEGF, and the formation of tubule-like networks. However, BMP2 did not activate the angiogenic processes. Osteogenesis was also significantly enhanced by the release of Co ions as well as BMP2, characterized by higher expression of osteogenic genes (OPN, ALP, BSP, and OCN), and OCN protein secretion. An in vivo study on the designed scaffolds implanted in rat calvarium defect demonstrated significantly enhanced bone formation, evidenced by new bone volume and bone density, due to the release of BMP2 and Co ions. This is the first study using Co ions as an angiogenic element together with the osteogenic factor BMP2 within scaffolds, and the results demonstrated the possible synergistic role of Co ions with BMP2 in the bone regeneration process, suggesting a novel potential therapeutic scaffold system. STATEMENT OF SIGNIFICANCE This is the first report that utilizes Co ion as a pro-angiogenic factor in concert with osteogenic factor BMP-2 in the fine-tuned core-shell hydrogel fiber scaffolds, and ultimately achieves osteo/angiogenesis of MSCs and bone regeneration through the sequential delivery of both biofactors. This novel approach facilitates a new class of therapeutic scaffolds, aiming at successful bone regeneration with the help of angiogenesis.