Nanofunctionalized Microparticles for Glucose Delivery in Three-Dimensional Cell Assemblies.

Three-dimensional (3D) cell assemblies, such as multicellular spheroids, can be powerful biological tools to closely mimic the complexity of cell-cell and cell-matrix interactions in a native-like microenvironment. However, potential applications of large spheroids are limited by the insufficient diffusion of oxygen and nutrients through the spheroids and, thus, result in the formation of a necrotic core. To overcome this drawback, we present a new strategy based on nanoparticle-coated microparticles. In this study, microparticles function as synthetic centers to regulate the diffusion of small molecules, such as oxygen and nutrients, within human mesenchymal stem cell (hMSC) spheroids. The nanoparticle coating on the microparticle surface acts as a nutrient reservoir to release glucose locally within the spheroids. We first coated the surface of the poly(lactic-co-glycolic acid) (PLGA) microparticles with mesoporous silica nanoparticles (MSNs) based on electrostatic interactions and then formed cell-nanofunctionalized microparticle spheroids. Next, we investigated the stability of the MSN coating on the microparticles' surface during 14 days of incubation in cell culture medium at 37 °C. Then, we evaluated the influence of MSN-coated PLGA microparticles on spheroid aggregation and cell viability. Our results showed the formation of homogeneous spheroids with good cell viability. As a proof of concept, fluorescently labeled glucose (2-NBD glucose) was loaded into the MSNs at different concentrations, and the release behavior was monitored. For cell culture studies, glucose was loaded into the MSNs coated onto the PLGA microparticles to sustain local nutrient release within the hMSC spheroids. In vitro results demonstrated that the local delivery of glucose from MSNs enhanced the cell viability in spheroids during a short-term hypoxic culture. Taken together, the newly developed nanofunctionalized microparticle-based delivery system may offer a versatile platform for local delivery of small molecules within 3D cellular assemblies and, thus, improve cell viability in spheroids.

Mini-bones: miniaturized bone in vitro models.

In bone tissue engineering (TE) and regeneration, miniaturized, (sub)millimeter-sized bone models have become a popular trend since they bring about physiological biomimicry, precise orchestration of concurrent stimuli, and compatibility with high-throughput setups and high-content imaging. They also allow efficient use of cells, reagents, materials, and energy. In this review, we describe the state of the art of miniaturized in vitro bone models, or 'mini-bones', describing these models based on their characteristics of (multi)cellularity and engineered extracellular matrix (ECM), and elaborating on miniaturization approaches and fabrication techniques. We analyze the performance of 'mini-bone' models according to their applications for studying basic bone biology or as regeneration models, disease models, and screening platforms, and provide an outlook on future trends, challenges, and opportunities.

Microfluidically Aligned Collagen to Maintain the Phenotype of Tenocytes In Vitro.

Tendon is a highly organized tissue that transmits forces between muscle and bone. The architecture of the extracellular matrix of tendon, predominantly from collagen type I, is important for maintaining tenocyte phenotype and function. Therefore, in repair and regeneration of damaged and diseased tendon tissue, it is crucial to restore the aligned arrangement of the collagen type I fibers of the original matrix. To this end, a novel, user-friendly microfluidic piggyback platform is developed allowing the controlled patterned formation and alignment of collagen fibers simply on the bottom of culture dishes. Rat tenocytes cultured on the micropatterns of aligned fibrous collagen exhibit a more elongated morphology. The cells also show an increased expression of tenogenic markers at the gene and protein level compared to tenocytes cultured on tissue culture plastic or non-fibrillar collagen coatings. Moreover, using imprinted polystyrene replicas of aligned collagen fibers, this work shows that the fibrillar structure of collagen per se affects the tenocyte morphology, whereas the biochemical nature of collagen plays a prominent role in the expression of tenogenic markers. Beyond the controlled provision of aligned collagen, the microfluidic platform can aid in developing more physiologically relevant in vitro models of tendon and its regeneration.

An in vitro model system based on calcium- and phosphate ion-induced hMSC spheroid mineralization.

A challenge in regenerative medicine is creating the three-dimensional organic and inorganic in vitro microenvironment of bone, which would allow the study of musculoskeletal disorders and the generation of building blocks for bone regeneration. This study presents a microwell-based platform for creating spheroids of human mesenchymal stromal cells, which are then mineralized using ionic calcium and phosphate supplementation. The resulting mineralized spheroids promote an osteogenic gene expression profile through the influence of the spheroids' biophysical environment and inorganic signaling and require less calcium or phosphate to achieve mineralization compared to a monolayer culture. We found that mineralized spheroids represent an in vitro model for studying small molecule perturbations and extracellular mediated calcification. Furthermore, we demonstrate that understanding pathway signaling elicited by the spheroid environment allows mimicking these pathways in traditional monolayer culture, enabling similar rapid mineralization events. In sum, this study demonstrates the rapid generation and employment of a mineralized cell model system for regenerative medicine applications.

Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids.

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection.

Assessment of Cell-Material Interactions in Three Dimensions through Dispersed Coaggregation of Microsized Biomaterials into Tissue Spheroids.

In biomaterials R&D, conventional monolayer cell culture on flat/planar material samples, such as films, is still commonly employed at early stages of the assessment of interactions of cells with candidate materials considered for a biomedical application. In this feasibility study, an approach for the assessment of 3D cell-material interactions through dispersed coaggregation of microparticles from biomaterials into tissue spheroids is presented. Biomaterial microparticles can be created comparatively quickly and easily, allow the miniaturization of the assessment platform, and enable an unhindered remodeling of the dynamic cell-biomaterial system at any time. The aggregation of the microsized biomaterials and the cells is supported by low-attachment round-bottom microwells from thin polymer films arranged in densely packed arrays. The study is conducted by the example of MG63 osteoblast-like and human mesenchymal stem/stromal cells, and a small library of model microbiomaterials related to bone repair and regeneration. For the proof of concept, example interactions including cell adhesion to the material, the hybrid spheroids' morphology, size, and shape, material-associated cell death, cell metabolic activity, cell proliferation, and (osteogenic) differentiation are investigated. The cells in the spheroids are shown to respond to differences in the microbiomaterials' properties, their amounts, and the duration of interaction with them.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Biomaterial-induced pathway modulation for bone regeneration.

Embryogenic developmental processes involve a tightly controlled regulation between mechanical forces and biochemical cues such as growth factors, matrix proteins, and cytokines. This interplay remains essential in the mature body, with aberrant pathway signaling leading to abnormalities such as atherosclerosis in the cardiovascular system, inflammation in tendon tissue, or osteoporosis in the bone. The aim of bone regenerative strategies is to develop tools and procedures that will harness the body's own self-repair ability in order to successfully regenerate even very large and complex bone defects and restore normal function. To achieve this, understanding pathways that govern processes of progenitor differentiation towards the osteogenic lineages, their phenotypical maintenance, and the construction of functional bone tissue is imperative to subsequently develop regenerative therapies that mimic these processes. While a body of literature exists that describes how biochemical stimuli guide cell behavior in the culture dish, due to the lack of an appropriate mechanical environment, these signals are often insufficient or inappropriate for achieving a desirable response in the body. Moreover, bone regenerative therapies rarely rely on a biochemical stimulus, such as a growth factor alone, and instead often comprise a carrier biomaterial that introduces a very different microenvironment from that of a cell culture dish. Therefore, in this review, we discuss which biomaterials elicit or influence pathways relevant for bone regeneration and describe mechanisms behind these effects, with the aim to inspire the development of novel, more effective bone regenerative therapies.

Calcium Phosphate-Coated and Strontium-Incorporated Mesoporous Silica Nanoparticles Can Effectively Induce Osteogenic Stem Cell Differentiation.

Ceramic (nano)materials are promising materials for bone regeneration applications. The addition of bioinorganics such as strontium (Sr) and zinc (Zn) is a popular approach to further improve their biological performance. However, control over ion delivery is important to prevent off-target effects. Mesoporous silica nanoparticles (MSNs) are popular nanomaterials that can be designed to incorporate and controllably deliver multiple ions to steer specific regenerative processes. In this work, MSNs loaded with Sr (MSNSr ) and surface coated with a pH-sensitive calcium phosphate (MSNSr -CaP) or calcium phosphate zinc layer (MSNSr -CaZnP) are developed. The ability of the MSNs to promote osteogenesis in human mesenchymal stromal cells (hMSCs) under basic cell culture conditions is explored and compared to ion administration directly to the cell culture media. Here, it is shown that MSN-CaPs can effectively induce alkaline phosphatase (ALP) levels and osteogenic gene expression in the absence of other osteogenic stimulants, where an improved effect is observed for MSNs surface coated with multiple ions. Moreover, comparatively lower ion doses are needed when using MSNs as delivery vehicles compared to direct ion administration in the medium. In summary, the MSNs developed here represent promising vehicles to deliver (multiple) bioinorganics and promote hMSC osteogenesis in basic conditions.

Chips for Biomaterials and Biomaterials for Chips: Recent Advances at the Interface between Microfabrication and Biomaterials Research.

In recent years, the use of microfabrication techniques has allowed biomaterials studies which were originally carried out at larger length scales to be miniaturized as so-called "on-chip" experiments. These miniaturized experiments have a range of advantages which have led to an increase in their popularity. A range of biomaterial shapes and compositions are synthesized or manufactured on chip. Moreover, chips are developed to investigate specific aspects of interactions between biomaterials and biological systems. Finally, biomaterials are used in microfabricated devices to replicate the physiological microenvironment in studies using so-called "organ-on-chip," "tissue-on-chip" or "disease-on-chip" models, which can reduce the use of animal models with their inherent high cost and ethical issues, and due to the possible use of human cells can increase the translation of research from lab to clinic. This review gives an overview of recent developments at the interface between microfabrication and biomaterials science, and indicates potential future directions that the field may take. In particular, a trend toward increased scale and automation is apparent, allowing both industrial production of micron-scale biomaterials and high-throughput screening of the interaction of diverse materials libraries with cells and bioengineered tissues and organs.

Transcript-Activated Coatings on Titanium Mediate Cellular Osteogenesis for Enhanced Osteointegration.

Osteointegration is one of the most important factors for implant success. Several biomolecules have been used as part of drug delivery systems to improve implant integration into the surrounding bone tissue. Chemically modified mRNA (cmRNA) is a new form of therapeutic that has been used to induce bone healing. Combined with biomaterials, cmRNA can be used to develop transcript-activated matrices for local protein production with osteoinductive potential. In this study, we aimed to utilize this technology to create bone morphogenetic protein 2 (BMP2) transcript-activated coatings for titanium (Ti) implants. Therefore, different coating methodologies as well as cmRNA incorporation strategies were evaluated. Three different biocompatible biomaterials were used for the coating of Ti, namely, poly-d,l-lactic acid (PDLLA), fibrin, and fibrinogen. cmRNA-coated Ti disks were assayed for transfection efficiency, cmRNA release, cell viability and proliferation, and osteogenic activity in vitro. We found that cmRNA release was significantly delayed in Ti surfaces previously coated with biomaterials. Consequently, the transfection efficiency was greatly improved. PDLLA coating improved the transfection efficiency in a concentration-dependent manner. Lower PDLLA concentration used for the coating of Ti resulted in higher transfection efficiency. Fibrin and fibrinogen coatings showed even higher transfection efficiencies compared to all PDLLA concentrations. In those disks, not only the expression was up to 24-fold higher but also the peak of maximal expression was delayed from 24 h to 5 days, and the duration of expression was also extended until 7 days post-transfection. For fibrin, higher transfection efficiencies were obtained in the coatings with the lowest thrombin amounts. Accordingly, fibrinogen coatings gave the best results in terms of cmRNA transfection. All biomaterial-coated Ti surfaces showed improved cell viability and proliferation, though this was more noticeable in the fibrinogen-coated disks. The latter was also the only coating to support significant amounts of BMP2 produced by C2C12 cells in vitro. Osteogenesis was confirmed using BMP2 cmRNA fibrinogen-coated Ti disks, and it was dependent of the cmRNA amount present. Alkaline phosphatase (ALP) activity of C2C12 increased when using fibrinogen coatings containing 250 ng of cmRNA or more. Similarly, mineralization was also observed that increased with increasing cmRNA concentration. Overall, our results support fibrinogen as an optimal material to deliver cmRNA from titanium-coated surfaces.

Stimulatory effect of cobalt ions incorporated into calcium phosphate coatings on neovascularization in an in vivo intramuscular model in goats.

UNLABELLED: Rapid vascularization of bone graft substitutes upon implantation is one of the most important challenges to overcome in order to achieve successful regeneration of large, critical-size bone defects. One strategy for stimulating vascularization during the regeneration process is to create a hypoxic microenvironment by either directly lowering the local oxygen tension, or by applying hypoxia-mimicking factors. Cells compensate for the hypoxic condition by releasing angiogenic factors leading to new blood vessel formation. In the present study, we explored the potential of cobalt ions (Co(2+)), known chemical mimickers of hypoxia, to stimulate vascularization within a bone graft substitute in vivo. To this end, Co(2+) ions were incorporated into calcium phosphate (CaPs) coatings deposited on poly(lactic acid) (PLA) particles with their effect on the formation of new blood vessels studied upon intramuscular implantation in goats. PLA particles and CaP-coated particles without Co(2+) ions served as controls. Pathological scoring of the inflammatory response following a 12-week implantation period showed no significant differences between the four types of materials. Based on histological and immunohistochemical analyses, both blood vessel area and number of blood vessels in CaP-coated PLA particles containing Co(2+) were higher than in the uncoated PLA particles and CaP-coated PLA particles without Co(2+). Analysis of blood vessel size distribution indicated abundant formation of small blood vessels in all the samples, while large blood vessels were predominantly found in PLA particles coated with CaP containing Co(2+) ions. The results of this study support the use of CaPs containing Co(2+) ions to enhance vascularization in vivo. STATEMENT OF SIGNIFICANCE: In this work, we have investigated the potential of cobalt ions, incorporated into thin calcium phosphate (CaP) coatings that were deposited on particles of poly(lactic acid) (PLA), to induce neovascularization in vivo. Qualitative and quantitative histological and immunohistochemical analyses showed that both the number of blood vessels and the total blood vessel area were higher in CaP-coated PLA particles containing cobalt ions as compared to the uncoated PLA particles and CaP-coated PLA particles without the metallic additive. Furthermore, a wider distribution of blood vessel sizes, varying from very small to large vessels was specifically observed in samples containing cobalt ions. This in vivo study will significantly contribute to the existing knowledge on the use of bioinorganics, which are simple and inexpensive inorganic factors that can be used to control relevant biological process during tissue regeneration, such as vascularization. As such, we are convinced that this manuscript will be of interest to the readers of Acta Biomaterialia.

Monolithic calcium phosphate/poly(lactic acid) composite versus calcium phosphate-coated poly(lactic acid) for support of osteogenic differentiation of human mesenchymal stromal cells.

Calcium phosphates (CaPs), extensively used synthetic bone graft substitutes, are often combined with other materials with the aim to overcome issues related to poor mechanical properties of most CaP ceramics. Thin ceramic coatings on metallic implants and polymer-ceramic composites are examples of such hybrid materials. Both the properties of the CaP used and the method of incorporation into a hybrid structure are determinant for the bioactivity of the final construct. In the present study, a monolithic composite comprising nano-sized CaP and poly(lactic acid) (PLA) and a CaP-coated PLA were comparatively investigated for their ability to support proliferation and osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells (hMSCs). Both, the PLA/CaP composite, produced using physical mixing and extrusion and CaP-coated PLA, resulting from a biomimetic coating process at near-physiological conditions, supported proliferation of hMSCs with highest rates at PLA/CaP composite. Enzymatic alkaline phosphatase activity as well as the mRNA expression of bone morphogenetic protein-2, osteopontin and osteocalcin were higher on the composite and coated polymer as compared to the PLA control, while no significant differences were observed between the two methods of combining CaP and PLA. The results of this study confirmed the importance of CaP in osteogenic differentiation while the exact properties and the method of incorporation into the hybrid material played a less prominent role.

Bone regeneration performance of surface-treated porous titanium.

The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid-alkali (AcAl), alkali-acid-heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial.

Enzymatic pH control for biomimetic deposition of calcium phosphate coatings.

The current study examines the enzymatic decomposition of urea into carbon dioxide and ammonia as a means to increase the pH during biomimetic deposition of calcium phosphate (CaP) onto implant surfaces. The kinetics of the enzymatically induced pH increase were studied by monitoring pH, calcium concentration and conductivity of the aqueous solutions as a function of time, urease concentration and initial concentrations of calcium and phosphate ions. Cryogenic transmission electron microscopy was used to study the process of homogeneous CaP precipitation in solution, whereas CaP deposition on conventional acid-etched titanium and micropatterned polystyrene (PS) surfaces was studied using scanning electron microscopy. The data presented in this study confirm that the substrate-enzyme combination urea-urease offers strong control over the rate of pH increase by varying the concentrations of precursor salts and urease. Formation of biomimetic CaP coatings was shown to proceed via formation of ionic polymeric assemblies of prenucleation complexes. The process of deposition and corresponding coating morphology was strongly dependent on the concentration of calcium, phosphate and urease. Finally, it was shown that the substrate-enzyme combination urea-urease allowed for spatial distribution of CaP crystals along the grooves of micropatterned PS surfaces at low concentrations of calcium, phosphate and urease, stressing the sensitivity of the presented method.

Molecular mechanisms of biomaterial-driven osteogenic differentiation in human mesenchymal stromal cells.

Calcium phosphate (CaP) based ceramics are used as bone graft substitutes in the treatment of bone defects. The physico-chemical properties of these materials determine their bioactivity, meaning that molecular and cellular responses in the body will be tuned accordingly. In a previous study, we compared two porous CaP ceramics, hydroxyapatite (HA) and ß-tricalcium phosphate (TCP), which, among other properties, differ in their degradation behaviour in vitro and in vivo, and we demonstrated that the more degradable ß-TCP induced more bone formation in a heterotopic model in sheep. This is correlated to in vitro data, where human bone marrow derived mesenchymal stromal cells (MSC) exhibited higher expression of osteogenic differentiation markers, such as osteopontin, osteocalcin and bone sialoprotein, when cultured in ß-TCP than in HA. More recently, we also showed that this effect could be mimicked in vitro by exposure of MSC to high concentrations of calcium ions (Ca(2+)). To further correlate surface physico-chemical dynamics of HA and ß-TCP ceramics with the molecular response of MSC, we followed Ca(2+) release and surface changes in time as well as cell attachment and osteogenic differentiation of MSC on these ceramics. Within 24 hours, we observed differences in cell morphology, with MSC cultured in ß-TCP displaying more pronounced attachment and spreading than cells cultured in HA. In the same time frame, ß-TCP induced expression of G-protein coupled receptor (GPCR) 5A and regulator of G-protein signaling 2, revealed by DNA microarray analysis. These genes, associated with the protein kinase A and GPCR signaling pathways, may herald the earliest response of MSC to bone-inducing ceramics.

Subcutaneous tissue response and osteogenic performance of calcium phosphate nanoparticle-enriched hydrogels in the tibial medullary cavity of guinea pigs.

In the current study, oligo(poly(ethylene glycol) fumarate) (OPF)-based hydrogels were tested for the first time as injectable bone substitute materials. The primary feature of the material design was the incorporation of calcium phosphate (CaP) nanoparticles within the polymeric matrix in order to compare the soft tissue response and bone-forming capacity of plain OPF hydrogels with CaP-enriched OPF hydrogel composites. To that end, pre-set scaffolds were implanted subcutaneously, whereas flowable polymeric precursor solutions were injected in a tibial ablation model in guinea pigs. After 8 weeks of implantation, histological and histomorphometrical evaluation of the subcutaneous scaffolds confirmed the biocompatibility of both types of hydrogels. Nevertheless, OPF hydrogels presented a loose structure, massive cellular infiltration and extensive material degradation compared to OPF-CaP hydrogels that were more compact. Microcomputed tomography and histological and histomorphometrical analyses showed comparable amounts of new trabecular bone in all tibias and some material remnants in the medial and distal regions. Particularly, highly calcified areas were observed in the distal region of OPF-CaP-treated tibias, which indicate a heterogeneous distribution of the mineral phase throughout the hydrogel matrix. This phenomenon can be attributed to either hindered gelation under highly perfused in vivo conditions or a faster degradation rate of the polymeric hydrogel matrix compared to the nanostructured mineral phase, resulting in loss of entrapment of the CaP nanoparticles and subsequent sedimentation.

Monolithic and assembled polymer-ceramic composites for bone regeneration.

The rationale for the use of polymer-ceramic composites for bone regeneration stems from the natural composition of bone, with collagen type I and biological apatite as the main organic and inorganic constituents, respectively. In the present study composite materials of PolyActive™ (PA), a poly(ethylene oxide terephthalate)/poly(butylene terephtalate) co-polymer, and hydroxyapatite (HA) at a weight ratio of 85:15 were prepared by rapid prototyping (RP) using two routes. In the first approach pre-extruded composite filaments of PA-HA were processed using three-dimensional fibre deposition (3DF) (conventional composite scaffolds). In the second approach PA scaffolds were fabricated using 3DF and combined with HA pillars produced inside stereolithographic moulds that fitted inside the pores of the PA three-dimensional structure (assembled composite scaffolds). Analysis of calcium and phosphate release in a simulated physiological solution, not containing calcium or phosphate, revealed significantly higher values for the HA pillars compared with other scaffolds. Release in simulated body fluid saturated with respect to HA did not show significant differences among the different scaffolds. Human mesenchymal stromal cells were cultured on polymer (3DF), conventional composite (3DF-HA) and assembled composite (HA assembled in 3DF) scaffolds and assessed for morphology, metabolic activity, DNA amount and gene expression of osteogenic markers using real time quantitative polymerase chain reaction (PCR). Scanning electron microscopy images showed that the cells attached to and infiltrated all the scaffolds. Assembled composites had a higher metabolic activity compared with 3DF-HA scaffolds while no significant differences were observed in DNA amounts. Gene expression of osteopontin in the assembled composite was significantly higher compared with the conventional composites. The strategy of composite fabrication by assembly appears to be a promising alternative to the conventional composite fabrication route for scaffolds for bone regeneration.