Advances in three‐dimensional bioprinting of bone: Progress and challenges

Several attempts have been made to engineer a viable three‐dimensional (3D) bone tissue equivalent using conventional tissue engineering strategies, but with limited clinical success. Using 3D bioprinting technology, scientists have developed functional prototypes of clinically relevant and mechanically robust bone with a functional bone marrow. Although the field is in its infancy, it has shown immense potential in the field of bone tissue engineering by re‐establishing the 3D dynamic micro‐environment of the native bone. Inspite of their in vitro success, maintaining the viability and differentiation potential of such cell‐laden constructs overtime, and their subsequent preclinical testing in terms of stability, mechanical loading, immune responses, and osseointegrative potential still needs to be explored. Progress is slow due to several challenges such as but not limited to the choice of ink used for cell encapsulation, optimal cell source, bioprinting method suitable for replicating the heterogeneous tissues and organs, and so on. Here, we summarize the recent advancements in bioprinting of bone, their limitations, challenges, and strategies for future improvisations. The generated knowledge will provide deep insights on our current understanding of the cellular interactions with the hydrogel matrices and help to unravel new methodologies for facilitating precisely regulated stem cell behaviour.     

Cell‐free artificial implants of electrospun fibres in a three‐dimensional gelatin matrix support sciatic nerve regeneration in vivo

Surgical repair of larger peripheral nerve lesions requires the use of autologous nerve grafts. At present, clinical alternatives to avoid nerve transplantation consist of empty tubes, which are only suitable for the repair over short distances and have limited success. We developed a cell‐free, three‐dimensional scaffold for axonal guidance in long‐distance nerve repair. Sub‐micron scale fibres of biodegradable poly‐ε‐caprolactone (PCL) and collagen/PCL (c/PCL) blends were incorporated in a gelatin matrix and inserted in collagen tubes. The conduits were tested by replacing 15‐mm‐long segments of rat sciatic nerves in vivo. Biocompatibility of the implants and nerve regeneration were assessed histologically, with electromyography and with behavioural tests for motor functions. Functional repair was achieved in all animals with autologous transplants, in 12 of 13 rats that received artificial implants with an internal structure and in half of the animals with empty nerve conduits. In rats with implants containing c/PCL fibres, the extent of recovery (compound muscle action potentials, motor functions of the hind limbs) was superior to animals that had received empty implants, but not as good as with autologous nerve transplantation. Schwann cell migration and axonal regeneration were observed in all artificial implants, and muscular atrophy was reduced in comparison with animals that had received no implants. The present design represents a significant step towards cell‐free, artificial nerve bridges that can replace autologous nerve transplants in the clinic. Copyright © 2017 John Wiley & Sons, Ltd.     

In situ handheld three‐dimensional bioprinting for cartilage regeneration

Articular cartilage injuries experienced at an early age can lead to the development of osteoarthritis later in life. In situ three‐dimensional (3D) printing is an exciting and innovative biofabrication technology that enables the surgeon to deliver tissue‐engineering techniques at the time and location of need. We have created a hand‐held 3D printing device (biopen) that allows the simultaneous coaxial extrusion of bioscaffold and cultured cells directly into the cartilage defect in vivo in a single‐session surgery. This pilot study assessed the ability of the biopen to repair a full‐thickness chondral defect and the early outcomes in cartilage regeneration, and compared these results with other treatments in a large animal model. A standardized critical‐sized full‐thickness chondral defect was created in the weight‐bearing surface of the lateral and medial condyles of both femurs of six sheep. Each defect was treated with one of the following treatments: (i) hand‐held in situ 3D printed bioscaffold using the biopen (HH group), (ii) preconstructed bench‐based printed bioscaffolds (BB group), (iii) microfractures (MF group) or (iv) untreated (control, C group). At 8 weeks after surgery, macroscopic, microscopic and biomechanical tests were performed. Surgical 3D bioprinting was performed in all animals without any intra‐ or postoperative complication. The HH biopen allowed early cartilage regeneration. The results of this study show that real‐time, in vivo bioprinting with cells and scaffold is a feasible means of delivering a regenerative medicine strategy in a large animal model to regenerate articular cartilage.     

Extracellular matrix deposition and scaffold biodegradation in an in vitro three‐dimensional model of bone by X‐ray computed microtomography

The development of an in vitro model of bone and the optimization of tools for determining the biological processes occurring during bone repair remains a major goal in the field of bone tissue engineering. Recently, a model based on a three‐dimensional co‐culture of osteoblasts and osteoclast precursors in Skelite^TM scaffolds was developed. Although induction of osteoblast and osteoclast differentiation was observed, a complete evaluation of bone deposition and biodegradation processes was missing due to technical limitations. In the current study, both X‐ray computed microtomography and histological analysis were used to monitor these two key biological processes in the same in vitro model. Either osteoblasts or a combination of osteoblasts and osteoclasts were seeded on Skelite^TM scaffolds. Scaffold biodegradation and increased bone deposition together with a more organized extracellular matrix were observed in the co‐cultures, highlighting the role of osteoclasts in the determination and regulation of bone deposition. Results confirmed the potential and relevance of co‐culturing osteoblasts and osteoclasts to resemble native tissue. The combination of X‐ray computed microtomography and histology presented in this study could be useful in future studies for the validation and development of new in vitro culture systems for bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

An additive manufacturing‐based PCL–alginate–chondrocyte bioprinted scaffold for cartilage tissue engineering

Regenerative medicine is targeted to improve, restore or replace damaged tissues or organs using a combination of cells, materials and growth factors. Both tissue engineering and developmental biology currently deal with the process of tissue self‐assembly and extracellular matrix (ECM) deposition. In this investigation, additive manufacturing (AM) with a multihead deposition system (MHDS) was used to fabricate three‐dimensional (3D) cell‐printed scaffolds using layer‐by‐layer (LBL) deposition of polycaprolactone (PCL) and chondrocyte cell‐encapsulated alginate hydrogel. Appropriate cell dispensing conditions and optimum alginate concentrations for maintaining cell viability were determined. In vitro cell‐based biochemical assays were performed to determine glycosaminoglycans (GAGs), DNA and total collagen contents from different PCL–alginate gel constructs. PCL–alginate gels containing transforming growth factor‐β (TGFβ) showed higher ECM formation. The 3D cell‐printed scaffolds of PCL–alginate gel were implanted in the dorsal subcutaneous spaces of female nude mice. Histochemical [Alcian blue and haematoxylin and eosin (H&E) staining] and immunohistochemical (type II collagen) analyses of the retrieved implants after 4 weeks revealed enhanced cartilage tissue and type II collagen fibril formation in the PCL–alginate gel (+TGFβ) hybrid scaffold. In conclusion, we present an innovative cell‐printed scaffold for cartilage regeneration fabricated by an advanced bioprinting technology. Copyright © 2013 John Wiley & Sons, Ltd.     

A novel three‐dimensional scaffold for regenerative endodontics: materials and biological characterizations

An electrospun nanocomposite fibrous material holds promise as a scaffold, as well as a drug‐delivery device to aid in root maturogenesis and the regeneration of the pulp–dentine complex. A novel three‐dimensional (3D) nanocomposite scaffold composed of polydioxanone (PDS II®) and halloysite nanotubes (HNTs) was designed and fabricated by electrospinning. Morphology, structure, mechanical properties and cell compatibility studies were carried out to evaluate the effects of HNTs incorporation (0.5–10 wt% relative to PDS w/w). Overall, a 3D porous network was seen in the different fabricated electrospun scaffolds, regardless of the HNT content. The incorporation of HNTs at 10 wt% led to a significant (p < 0.0001) fibre diameter increase and a reduction in scaffold strength. Moreover, PDS–HNTs scaffolds supported the attachment and proliferation of human‐derived pulp fibroblast cells. Quantitative proliferation assay performed with human dental pulp‐derived cells as a function of nanotubes concentration indicated that the HNTs exhibit a high level of biocompatibility, rendering them good candidates for the potential encapsulation of distinct bioactive molecules. Collectively, the reported data support the conclusion that PDS–HNTs nanocomposite fibrous structures hold potential in the development of a bioactive scaffold for regenerative endodontics. Copyright © 2013 John Wiley & Sons, Ltd.     

Tissue‐engineered trachea from a 3D‐printed scaffold enhances whole‐segment tracheal repair in a goat model

Traditional treatment therapies for tracheal stenosis often cause severe post‐operative complications. To solve the current difficulties, novel and more suitable long‐term treatments are needed. A whole‐segment tissue‐engineered trachea (TET) representing the native goat trachea was 3D printed using a poly(caprolactone) (PCL) scaffold engineered with autologous auricular cartilage cells. The TET underwent mechanical analysis followed by in vivo implantations in order to evaluate the clinical feasibility and potential. The 3D‐printed scaffolds were successfully cellularized, as observed by scanning electron microscopy. Mechanical force compression studies revealed that both PCL scaffolds and TETs have a more robust compressive strength than does the native trachea. In vivo implantation of TETs in the experimental group resulted in significantly higher mean post‐operative survival times, 65.00 ± 24.01 days (n = 5), when compared with the control group, which received autologous trachea grafts, 17.60 ± 3.51 days (n = 5). Although tracheal narrowing was confirmed by bronchoscopy and computed tomography examination in the experimental group, tissue necrosis was only observed in the control group. Furthermore, an encouraging epithelial‐like tissue formation was observed in the TETs after transplantation. This large animal study provides potential preclinical evidence around the employment of an orthotopic transplantation of a whole 3D‐printed TET.     

Hyaline cartilage next generation implants from adipose‐tissue–derived mesenchymal stem cells: Comparative study on 3D‐printed polycaprolactone scaffold patterns

We used additive manufacturing to fabricate 3D‐printed polycaprolactone scaffolds of different geometry topologies and porosities. We present a comparative analysis of hyaline cartilage development from adipose‐tissue–derived mesenchymal stem cells (ADMSCs) on three different, newly designed scaffold geometry patterns. The first scaffold design (MESO) was based on a rectilinear layer pattern. For the second pattern (RO45), we employed a 45° rotational layer loop. The design for the third scaffold (3DHC) was a three‐dimensional honeycomb‐like pattern with a hexagonal cellular distribution and small square shapes. We examined cell proliferation, colonization, and differentiation, in relation to the scaffold's structure, as well as to the mechanical properties of the final constructs. We gave emphasis on the scaffolds, both microarchitecture and macroarchitecture, for optimal and enhanced chondrogenic differentiation, as an important parameter, not well studied in the literature. Among the three patterns tested, RO45 was the most favourable for chondrogenic differentiation, whereas 3DHC better supported cell proliferation and scaffold penetration, exhibiting also the highest rate of increase onto the mechanical properties of the final construct. We conclude that by choosing the optimal scaffold architecture, the resulting properties of our cartilaginous constructs can better approximate those of the physiological cartilage.     

Controlled release of BMP‐2 from a sintered polymer scaffold enhances bone repair in a mouse calvarial defect model

Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP‐2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature‐sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP‐2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP‐2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP‐2 loaded into the scaffolds was released by the end of this time period. The released BMP‐2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP‐2‐loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP‐2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP‐2 delivery for bone‐regeneration applications. Copyright © 2012 John Wiley & Sons, Ltd.     

Recent advances in three‐dimensional bioprinting of stem cells

In spite of being a new field, three‐dimensional (3D) bioprinting has undergone rapid growth in the recent years. Bioprinting methods offer a unique opportunity for stem cell distribution, positioning, and differentiation at the microscale to make the differentiated architecture of any tissue while maintaining precision and control over the cellular microenvironment. Bioprinting introduces a wide array of approaches to modify stem cell fate. This review discusses these methodologies of 3D bioprinting stem cells. Fabricating a fully operational tissue or organ construct with a long life will be the most significant challenge of 3D bioprinting. Once this is achieved, a whole human organ can be fabricated for the defect place at the site of surgery.     

Local injection of lovastatin in biodegradable polyurethane scaffolds enhances bone regeneration in a critical‐sized segmental defect in rat femora

Statins, a class of naturally‐occurring compounds that inhibit HMG‐CoA reductase, are known to increase endogenous bone morphogenetic protein‐2 (BMP‐2) expression. Local administration of statins has been shown to stimulate fracture repair in in vivo animal experiments. However, the ability of statins to heal more challenging critical‐sized defects at the mid‐diaphyseal region in long bones has not been investigated. In this study, we examined the potential of injectable lovastatin microparticles combined with biodegradable polyurethane (PUR) scaffolds in preclinical animal models: metaphyseal small plug defects and diaphyseal segmental bone defects in rat femora. Sustained release of lovastatin from the lovastatin microparticles was achieved over 14 days. The released lovastatin was bioactive, as evidenced by its ability to stimulate BMP‐2 gene expression in osteoblastic cells. Micro‐computed tomography (CT) and histological examinations showed that lovastatin microparticles, injected into PUR scaffolds implanted in femoral plug defects, enhanced new bone formation. Furthermore, bi‐weekly multiple injections of lovastatin microparticles into PUR scaffolds implanted in critical‐sized femoral segmental defects resulted in increased new bone formation compared to the vehicle control. In addition, bridging of the defect with newly formed bone was observed in four of nine defects in the lovastatin microparticle treatment group, whereas none of the defects in the vehicle group showed bridging. These observations suggest that local delivery of lovastatin combined with PUR scaffold can be an effective approach for treatment of orthopaedic bone defects and that multiple injections of lovastatin may be useful for large defects. Copyright © 2012 John Wiley & Sons, Ltd.     

Loading of BMP‐2‐related peptide onto three‐dimensional nano‐hydroxyapatite scaffolds accelerates mineralization in critical‐sized cranial bone defects

Extrusion free‐forming, as a rapid prototyping technique, is extensively applied in fabricating ceramic material in bone tissue engineering. To improve the osteoinductivity of nano‐hydroxyapatite (nHA) scaffold fabricated by extrusion free‐forming, in this study, we incorporated a new peptide (P28) and optimized the superficial microstructure after shaping by controlling the sintering temperature. P28, a novel bone morphogenic protein 2 (BMP‐2)‐related peptide, was designed in this study. Analysis of the structure, physicochemical properties and release kinetics of P28 from nHA sintered at temperatures ranging from 1000 °C to 1400 °C revealed that nHA sintered at 1000 °C had higher porosity, preferable pore size and better capacity to control P28 release than that sintered at other temperatures. Moreover, the nHA scaffold sintered at 1000 °C with P28 showed improved adhesion, proliferation and osteogenic differentiation of MC3T3‐E1 cells compared with scaffolds lacking P28 or BMP‐2. In vivo, nHA scaffolds sintered at 1000 °C with P28 or BMP‐2 induced greater bone regeneration in critical‐sized rat cranial defects at 6 and 12 weeks post‐implantation compared with scaffolds lacking P28 or BMP‐2. Thus, nHA scaffolds sintered at 1000 °C and loaded with P28 may be excellent biomaterials for bone tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.     

Evaluation of a platelet lysate bilayered system for periodontal regeneration in a rat intrabony three‐wall periodontal defect

With currently available therapies, full regeneration of lost periodontal tissues after periodontitis cannot be achieved. In this study, a combined compartmentalized system was tested, composed of (a) a platelet lysate (PL)‐based construct, which was placed along the root aiming to regenerate the root cementum and periodontal ligament, and (b) a calcium phosphate cement composite incorporated with hyaluronic acid microspheres loaded with PL, aiming to promote the regeneration of alveolar bone. This bilayered system was assessed in a 3‐wall periodontal defect in Wistar rats. The periodontal healing and the inflammatory response of the materials were scored for a period up to 6 weeks after implantation. Furthermore, histomorphometrical measurements were performed to assess the epithelial downgrowth, the formation of alveolar bone, and the formation of new connective tissue attachment. Our data showed that the stabilization of platelet‐origin proteins on the root surface increased the overall periodontal healing score and restricted the formation of long epithelial junctions. Nevertheless, the faster degradation of the cement component with incorporated hyaluronic acid microspheres compromised the stability of the system, which hampered the periodontal regeneration. Overall, in this work, we proved the positive therapeutic effect of the immobilization of a PL‐based construct over the root surface in a combined compartmentalized system to assist predictable healing of functional periodontium. Therefore, after optimization of the hard tissue analogue, the system should be further elaborated in (pre)clinical validation studies.     

Local application of lactoferrin promotes bone regeneration in a rat critical‐sized calvarial defect model as demonstrated by micro‐CT and histological analysis

Lactoferrin is a multifunctional glycoprotein with therapeutic potential for bone tissue engineering. The aim of this study was to assess the efficacy of local application of lactoferrin on bone regeneration. Five‐millimetre critical‐sized defects were created over the right parietal bone in 64 Sprague–Dawley rats. The rats were randomized into four groups: group 1 (n  =  20) had empty defects; group 2 (n  =  20) had defects grafted with collagen gels (3 mg/ml); group 3 (n  =  20) had defects grafted with collagen gels impregnated with bovine lactoferrin (10 μg/gel); and group 4 (n  =  4) had sham surgeries (skin and periosteal incisions only). The rats were sacrificed at 4 or 12 weeks post‐operatively, and the calvaria were excised and evaluated with micro‐CT (Skyscan 1172) followed by histology. The bone volume fraction (BV/TV) was higher in lactoferrin‐treated animals at both timepoints, with groups 1, 2, 3 and 4 measuring 10.5  ±  1.1%, 8.6  ±  1.4%, 16.5  ±  0.6% and 24.27  ±  2.6%, respectively, at 4 weeks (P  <  0.05); and 12.2  ±  1.3%, 13.6  ±  1.5%, 21.9  ±  1.2% and 29.3  ±  0.8%, respectively, at 12 weeks (P  <  0.05). Histological analysis revealed that the newly formed bone within the calvarial defects of all groups was a mixture of woven and lamellar bone, with more bone in the group treated with lactoferrin at both timepoints. Our study demonstrated that local application of lactoferrin significantly increased bone regeneration in a rat critical‐sized calvarial defect model. The profound effect of lactoferrin on bone regeneration has therapeutic potential to improve the poor clinical outcomes associated with bony non‐union. LF In Vivo JTERM Authors Contributions. Copyright © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.     

Preparation of three‐dimensional macroporous chitosan–gelatin B microspheres and HepG2‐cell culture

Chitosan–gelatin B microspheres with an open, interconnected, highly macroporous (100–200 µm) structure were prepared via a three‐step protocol combining freeze‐drying with an electrostatic and ionic cross‐linking method. Saturated tripolyphosphate ethanol solution (85% ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure and to improve the biostability of the chitosan–gelatin B microspheres, with N‐(3‐dimethylaminopropyl)‐N′‐ethyl‐carbodiimide/N‐hydroxysuccinimide as a second crosslinking agent to react with gelatin A and fixed chitosan–gelatin B microspheres to attain improved biocompatibility. Water absorption of the three‐dimensional macroporous chitosan–gelatin B microspheres (3D‐P‐CGMs) was 12.84, with a porosity of 85.45%. In vitro lysozyme degradation after 1, 3, 5, 7, 10, 14, and 21 days showed improved biodegradation in the 3D‐P‐CGMs. The morphology of human hepatoma cell lines (HepG2 cells) cultured on the 3D‐P‐CGMs was spherical, unlike that of cells cultured under traditional two‐dimensional conditions. Scanning electron microscopy and paraffin sections were used to confirm the porous structure of the 3D‐P‐CGMs. HepG2 cells were able to migrate inside through the pore. Cell proliferation and levels of albumin and lactate dehydrogenase suggested that the 3D‐P‐CGMs could provide a larger specific surface area and an appropriate microenvironment for cell growth and survival. Hence, the 3D‐P‐CGMs are eminently suitable as macroporous scaffolds for cell cultures in tissue engineering and cell carrier studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a synthetic tissue engineered three‐dimensional printed bioceramic‐based bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro

Over the last decade there have been increasing efforts to develop three‐dimensional (3D) scaffolds for bone tissue engineering from bioactive ceramics with 3D printing emerging as a promising technology. The overall objective of the present study was to generate a tissue engineered synthetic bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro, thereby mimicking the advantageous properties of autogenous bone grafts and facilitating usage for reconstructing segmental discontinuity defects in vivo. To this end, 3D scaffolds were developed from a silica‐containing calcium alkali orthophosphate, using, first, a replica technique – the Schwartzwalder–Somers method – and, second, 3D printing, (i.e. rapid prototyping). The mechanical and physical scaffold properties and their potential to facilitate homogenous colonization by osteogenic cells and extracellular bone matrix formation throughout the porous scaffold architecture were examined. Osteoblastic cells were dynamically cultured for 7 days on both scaffold types with two different concentrations of 1.5 and 3 × 10⁹ cells/l. The amount of cells and bone matrix formed and osteogenic marker expression were evaluated using hard tissue histology, immunohistochemical and histomorphometric analysis. 3D‐printed scaffolds (RPS) exhibited more micropores, greater compressive strength and silica release. RPS seeded with 3 × 10⁹ cells/l displayed greatest cell and extracellular matrix formation, mineralization and osteocalcin expression. In conclusion, RPS displayed superior mechanical and biological properties and facilitated generating a tissue engineered synthetic bone graft in vitro, which mimics the advantageous properties of autogenous bone grafts, by containing homogenously distributed terminally differentiated osteoblasts and mineralizing bone matrix and therefore is suitable for subsequent in vivo implantation for regenerating segmental discontinuity bone defects. Copyright © 2016 John Wiley & Sons, Ltd.     

Tubular open‐porous β‐tricalcium phosphate polycaprolactone scaffolds as guiding structure for segmental bone defect regeneration in a novel sheep model

Large segmental bone defect reconstruction with sufficient functional restoration is one of the most demanding challenges in orthopaedic surgery. Available regenerative treatment options, as the vascularized bone graft transfer, the Masquelet technique or the Ilizarov distraction osteogenesis, are associated with specific indications and distinct limitations. As an alternative, a hollow cylindrical ceramic‐polymer composite scaffold (β‐tricalcium phosphate and poly‐lactid co‐ε‐ caprolactone), facilitating a strong surface guiding effect for tissue ingrowth (group 1; n = 6) was investigated here. In combination with an additional autologous, cancellous bone graft filling, the scaffold's ability to work as an open‐porous membrane to improve the defect healing process was analysed (group 2; n = 6). A novel model of a critical size (40 mm) tibia osteotomy defect stabilized with an external hybrid‐ring fixator, was established in sheep. Segmental defect regeneration and tissue organization in relation to the scaffold were analysed radiologically, (immune‐) histologically, and with second‐harmonic generation imaging 12 weeks after surgery. The scaffold's tubular shape and open‐porous structure controlled the collagen fibre orientation within the bone defect and guided the following mineralization process along the scaffold surface. In combination with the osteoinductive stimulus, a unilateral bony bridging of the critically sized defect was achieved in one third of the animals. The external hybrid‐ring fixator was appropriate for large segmental defect stabilization in sheep.     

Collagen density gradient on three‐dimensional printed poly(ε‐caprolactone) scaffolds for interface tissue engineering

The ability to engineer scaffolds that resemble the transition between tissues would be beneficial to improve repair of complex organs, but has yet to be achieved. In order to mimic tissue organization, such constructs should present continuous gradients of geometry, stiffness and biochemical composition. Although the introduction of rapid prototyping or additive manufacturing techniques allows deposition of heterogeneous layers and shape control, the creation of surface chemical gradients has not been explored on three‐dimensional (3D) scaffolds obtained through fused deposition modelling technique. Thus, the goal of this study was to introduce a gradient functionalization method in which a poly(ε‐caprolactone) surface was first aminolysed and subsequently covered with collagen via carbodiimide reaction. The 2D constructs were characterized for their amine and collagen contents, wettability, surface topography and biofunctionality. Finally, chemical gradients were created in 3D printed scaffolds with controlled geometry and porosity. The combination of additive manufacturing and surface modification is a viable tool for the fabrication of 3D constructs with controlled structural and chemical gradients. These constructs can be employed for mimicking continuous tissue gradients for interface tissue engineering.     

Design and biological functionality of a novel hybrid Ti‐6Al‐4V/hydrogel system for reconstruction of bone defects

We have designed a unique injectable bioactive hydrogel comprising of alginate, gelatin, and nanocrystalline hydroxyapatite and loaded with osteoblasts, with the ability to infiltrate into three‐dimensional Ti‐6Al‐4V scaffolds with interconnected porous architecture, fabricated by electron beam melting. A two‐step crosslinking process using the EDC/NHS and CaCl₂ was adopted and found to be effective in the fabrication of cell‐loaded hydrogel/Ti‐6Al‐4V scaffold system. This hybrid Ti‐6Al‐4V scaffold/hydrogel system was designed for the reconstruction of bone defects, which are difficult to heal in the absence of suitable support materials. The hybrid Ti‐6Al‐4V/hydrogel system favourably modulated the biological functions, namely, adhesion, proliferation, cell‐to‐cell, and cell‐material communication because of the presence of extracellular matrix‐like hydrogel in the interconnected porous structure of 3D printed Ti‐6Al‐4V scaffold. The hydrogel was cytocompatible, which was proven through live/dead assay, the expression level of prominent proteins for cell adhesion and cytoskeleton, including 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay. Furthermore, the high bone formation ability of the hydrogel was confirmed using alkaline phosphatase assay. A high equilibrium water content (~97%) in the hydrogel enables the delivery of cells and bioactive molecules, necessary for bone tissue growth. Although not studied, the presence of hydrogel in the pores of the scaffold can provide the space for the cell migration as well as vascularization through it, required for the effective exchange of nutrients. In conclusion, we underscore that the 3D‐printed Ti‐6Al‐4V scaffold‐loaded with bioactive hydrogel to treat the bone defects significantly impacted cellular functions and cell‐material interaction.     

Bone marrow‐on‐a‐chip: Long‐term culture of human haematopoietic stem cells in a three‐dimensional microfluidic environment

Multipotent haematopoietic stem and progenitor cells (HSPCs) are the source for all blood cell types. The bone marrow stem cell niche in which the HSPCs are maintained is known to be vital for their maintenance. Unfortunately, to date, no in vitro model exists that accurately mimics the aspects of the bone marrow niche and simultaneously allows the long‐term culture of HSPCs. In this study, a novel three‐dimensional coculture model is presented, based on a hydroxyapatite coated zirconium oxide scaffold, comprising of human mesenchymal stromal cells (MSCs) and cord blood derived HSPCs, enabling successful HSPC culture for a time span of 28 days within the microfluidic multiorgan chip. The HSPCs were found to stay in their primitive state (CD34⁺CD38^?) and capable of granulocyte, erythrocyte, macrophage, megakaryocyte colony formation. Furthermore, a microenvironment was formed bearing molecular and structural similarity to the in vivo bone marrow niche containing extracellular matrix and signalling molecules known to play an important role in HSPC homeostasis. Here, a novel human in vitro bone marrow model is presented for the first time, capable of long‐term culture of primitive HSPCs in a microfluidic environment.