Treatment of Critical Size Femoral Bone Defects with Biomimetic Hybrid Scaffolds of 3D Plotted Calcium Phosphate Cement and Mineralized Collagen Matrix.

To treat critical-size bone defects, composite materials and tissue-engineered bone grafts play important roles in bone repair materials. The purpose of this study was to investigate the bone regenerative potential of hybrid scaffolds consisting of macroporous calcium phosphate cement (CPC) and microporous mineralized collagen matrix (MCM). Hybrid scaffolds were synthetized by 3D plotting CPC and then filling with MCM (MCM-CPC group) and implanted into a 5 mm critical size femoral defect in rats. Defects left empty (control group) as well as defects treated with scaffolds made of CPC only (CPC group) and MCM only (MCM group) served as controls. Eight weeks after surgery, micro-computed tomography scans and histological analysis were performed to analyze the newly formed bone, the degree of defect healing and the activity of osteoclasts. Mechanical stability was tested by 3-point-bending of the explanted femora. Compared with the other groups, more newly formed bone was found within MCM-CPC scaffolds. The new bone tissue had a clamp-like structure which was fully connected to the hybrid scaffolds and thereby enhanced the biomechanical strength. Together, the biomimetic hybrid MCM-CPC scaffolds enhanced bone defect healing by improved osseointegration and their differentiated degradation provides spatial effects in the process of critical-bone defect healing.

Influence of Cu2+ on Osteoclast Formation and Activity In Vitro.

BACKGROUND: Copper-containing biomaterials are increasingly applied for bone regeneration due to their pro-angiogenetic, pro-osteogenetic and antimicrobial properties. Therefore, the effect of Cu2+ on osteoclasts, which play a major role in bone remodeling was studied in detail. METHODS: Human primary osteoclasts, differentiated from human monocytes were differentiated or cultivated in the presence of Cu2+. Osteoclast formation and activity were analyzed by measurement of osteoclast-specific enzyme activities, gene expression analysis and resorption assays. Furthermore, the glutathione levels of the cells were checked to evaluate oxidative stress induced by Cu2+. RESULTS: Up to 8 µM Cu2+ did not induce cytotoxic effects. Activity of tartrate-resistant acid phosphatase (TRAP) was significantly increased, while other osteoclast specific enzyme activities were not affected. However, gene expression of TRAP was not upregulated. Resorptive activity of osteoclasts towards dentin was not changed in the presence of 8 µM Cu2+ but decreased in the presence of extracellular bone matrix. When Cu2+ was added to mature osteoclasts TRAP activity was not increased and resorption decreased only moderately. The glutathione level of both differentiating and mature osteoclasts was significantly decreased in the presence of Cu2+. CONCLUSIONS: Differentiating and mature osteoclasts react differently to Cu2+. High TRAP activities are not necessarily related to high resorption.

Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration.

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization.

Triple Culture of Primary Human Osteoblasts, Osteoclasts and Osteocytes as an In Vitro Bone Model.

In vitro evaluation of bone graft materials is generally performed by analyzing the interaction with osteoblasts or osteoblast precursors. In vitro bone models comprising different cell species can give specific first information on the performance of those materials. In the present study, a 3D co-culture model was established comprising primary human osteoblasts, osteoclasts and osteocytes. Osteocytes were differentiated from osteoblasts embedded in collagen gels and were cultivated with osteoblast and osteoclasts seeded in patterns on a porous membrane. This experimental setup allowed paracrine signaling as well as separation of the different cell types for final analysis. After 7 days of co-culture, the three cell species showed their typical morphology and gene expression of typical markers like ALPL, BSPII, BLGAP, E11, PHEX, MEPE, RANKL, ACP5, CAII and CTSK. Furthermore, relevant enzyme activities for osteoblasts (ALP) and osteoclasts (TRAP, CTSK, CAII) were detected. Osteoclasts in triple culture showed downregulated TRAP (ACP5) and CAII expression and decreased TRAP activity. ALP and BSPII expression of osteoblasts in triple culture were upregulated. The expression of the osteocyte marker E11 (PDPN) was unchanged; however, osteocalcin (BGLAP) expression was considerably downregulated both in osteoblasts and osteocytes in triple cultures compared to the respective single cultures.

Anisotropic Chitosan Scaffolds Generated by Electrostatic Flocking Combined with Alginate Hydrogel Support Chondrogenic Differentiation.

The replacement of damaged or degenerated articular cartilage tissue remains a challenge, as this non-vascularized tissue has a very limited self-healing capacity. Therefore, tissue engineering (TE) of cartilage is a promising treatment option. Although significant progress has been made in recent years, there is still a lack of scaffolds that ensure the formation of functional cartilage tissue while meeting the mechanical requirements for chondrogenic TE. In this article, we report the application of flock technology, a common process in the modern textile industry, to produce flock scaffolds made of chitosan (a biodegradable and biocompatible biopolymer) for chondrogenic TE. By combining an alginate hydrogel with a chitosan flock scaffold (CFS+ALG), a fiber-reinforced hydrogel with anisotropic properties was developed to support chondrogenic differentiation of embedded human chondrocytes. Pure alginate hydrogels (ALG) and pure chitosan flock scaffolds (CFS) were studied as controls. Morphology of primary human chondrocytes analyzed by cLSM and SEM showed a round, chondrogenic phenotype in CFS+ALG and ALG after 21 days of differentiation, whereas chondrocytes on CFS formed spheroids. The compressive strength of CFS+ALG was higher than the compressive strength of ALG and CFS alone. Chondrocytes embedded in CFS+ALG showed gene expression of chondrogenic markers (COL II, COMP, ACAN), the highest collagen II/I ratio, and production of the typical extracellular matrix such as sGAG and collagen II. The combination of alginate hydrogel with chitosan flock scaffolds resulted in a scaffold with anisotropic structure, good mechanical properties, elasticity, and porosity that supported chondrogenic differentiation of inserted human chondrocytes and expression of chondrogenic markers and typical extracellular matrix.

Toward Biofabrication of Resorbable Implants Consisting of a Calcium Phosphate Cement and Fibrin-A Characterization In Vitro and In Vivo.

Cleft alveolar bone defects can be treated potentially with tissue engineered bone grafts. Herein, we developed novel biphasic bone constructs consisting of two clinically certified materials, a calcium phosphate cement (CPC) and a fibrin gel that were biofabricated using 3D plotting. The fibrin gel was loaded with mesenchymal stromal cells (MSC) derived from bone marrow. Firstly, the degradation of fibrin as well as the behavior of cells in the biphasic system were evaluated in vitro. Fibrin degraded quickly in presence of MSC. Our results showed that the plotted CPC structure acted slightly stabilizing for the fibrin gel. However, with passing time and fibrin degradation, MSC migrated to the CPC surface. Thus, the fibrin gel could be identified as cell delivery system. A pilot study in vivo was conducted in artificial craniofacial defects in Lewis rats. Ongoing bone formation could be evidenced over 12 weeks but the biphasic constructs were not completely osseous integrated. Nevertheless, our results show that the combination of 3D plotted CPC constructs and fibrin as suitable cell delivery system enables the fabrication of novel regenerative implants for the treatment of alveolar bone defects.

Effect of 'in air' freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds.

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen 'in air' and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of 'ready-to-use' TECs.

Characterization of Naturally Occurring Bioactive Factor Mixtures for Bone Regeneration.

In this study, the bone-regenerative potential of bioactive factors derived from adipose tissue, platelet-rich plasma (PRP) and conditioned medium from hypoxia-treated human telomerase immortalized bone-marrow-derived mesenchymal stem cells (hTERT-MSC) was investigated in vitro with the aim to develop cost-effective and efficient bone substitutes for optimized regeneration of bone defects. Adipose tissue was harvested from human donors undergoing reconstructive surgery, and adipose tissue extract (ATE) was prepared. Platelet lysates (PL) were produced by repeated freeze-thaw cycles of PRP, and hypoxia-conditioned medium (HCM) was obtained by culturing human telomerase immortalized bone-marrow-derived mesenchymal stromal cells for 5 days with 1% O2. Besides analysis by cytokine and angiogenesis arrays, ELISA was performed. Angiogenic potential was investigated in cocultures of bone-marrow-derived (BM)-MSC and human umbilical vein endothelial cells. Multiple angiogenic proteins and cytokines were detected in all growth factor mixtures. HCM and ATE contained high amounts of angiogenin and CCL2/MCP-1, whereas PL contained high amounts of IGFBP-1. Culturing cells with HCM and ATE significantly increased specific ALP activity of BM-MSC as well as tubule length and junctions of endothelial networks, indicating osteogenic and angiogenic stimulation. To achieve a synergism between chemoattractive potential and osteogenic and angiogenic differentiation capacity, a combination of different growth factors appears promising for potential clinical applications.

Naturally Drug-Loaded Chitin: Isolation and Applications.

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis. A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay.

Investigating the effect of sterilisation methods on the physical properties and cytocompatibility of methyl cellulose used in combination with alginate for 3D-bioplotting of chondrocytes.

For both the incorporation of cells and future therapeutic applications the sterility of a biomaterial must be ensured. However, common sterilisation techniques are intense and often negatively impact on material physicochemical attributes, which can affect its suitability for tissue engineering and 3D printing. In the present study four sterilisation methods, autoclave, supercritical CO2 (scCO2) treatment, UV- and gamma (γ) irradiation were evaluated regarding their impact on material properties and cellular responses. The investigations were performed on methyl cellulose (MC) as a component of an alginate/methyl cellulose (alg/MC) bioink, used for bioprinting embedded bovine primary chondrocytes (BPCs). In contrast to the autoclave, scCO2 and UV-treatments, the γ-irradiated MC resulted in a strong reduction in alg/MC viscosity and stability after extrusion which made this method unsuitable for precise bioprinting. Gel permeation chromatography analysis revealed a significant reduction in MC molecular mass only after γ-irradiation, which influenced MC chain mobility in the Ca2+-crosslinked alginate network as well as gel composition and microstructure. With regard to cell survival and proteoglycan matrix production, the results determined UV-irradiation and autoclaving as the best candidates for sterilisation. The scCO2-treatment of MC resulted in an unfavourable cell response indicating that this method needs careful optimisation prior to application for cell encapsulation. As proven by consistent FT-IR spectra, chemical alterations could be excluded as a cause for the differences seen between MC treatments on alg/MC behaviour. This investigation provides knowledge for the development of a clinically appropriate 3D-printing-based fabrication process to produce bioengineered tissue for cartilage regeneration.

In Vitro Co-culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels.

BACKGROUND: Osteocytes are the key regulator cells in bone tissue, affecting activity of both osteoblasts and osteoclasts. Current in vitro studies on osteocyte-osteoblast interaction are invariably performed with rodent cells, mostly murine cell lines, which diminishes the clinical relevance of the data. OBJECTIVE: The objective of the present study was to establish an in vitro co-culture system of osteoblasts and osteocytes, which is based solely on human primary cells. METHODS: Three different approaches for the generation of human primary osteocytes were compared: direct isolation of osteocytes from bone tissue by multistep digestion, long-time differentiation of human pre-osteoblasts embedded in collagen gels, and short time differentiation of mature human osteoblasts in collagen gels. Co-cultivation of mature osteoblasts with osteocytes, derived from the three different approaches was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoblasts on the basal side of a porous membrane, which allowed the separate gene expression analysis for osteocytes and osteoblasts. Fluorescence microscopic imaging and gene expression analysis were performed separately for osteocytes and osteoblasts. RESULTS: All examined approaches provided cells with typical osteocytic morphology, which expressed osteocyte markers E11, osteocalcin, phosphate regulating endopeptidase homolog, X-linked (PHEX), matrix extracellular phosphoglycoprotein (MEPE), sclerostin, and receptor activator of NF-κB Ligand (RANKL). Expression of osteocyte markers was not significantly changed in the presence of osteoblasts. In contrast, osteocalcin gene expression of osteoblasts was significantly upregulated in all examined co-cultures with differentiated osteocytes. Alkaline phosphatase (ALPL), bone sialoprotein II (BSPII), and RANKL expression of osteoblasts was not significantly changed in the co-culture. CONCLUSION: Interaction of osteoblasts and osteocytes can be monitored in an in vitro model, comprising solely primary human cells.

Influence of Regioselectively Sulfated Cellulose on in Vitro Vascularization of Biomimetic Bone Matrices.

Vascularization is essential for the regeneration of bone tissue within composite material. We measured the effect of regioselectively modified cellulose/hemicellulose as an additive for porous scaffolds of collagen/hydroxyapatite nanocomposite on the tubule formation of human vascular endothelial cells. Using a coculture of endothelial cells and fibroblasts, endothelial cells formed a network of tubules within an incubation time of 14 to 24 days. A cellulose sulfate with irregular sulfation pattern along the polysaccharide backbone (13-TACS-01) led to an additional increase in vascular endothelial growth factor (VEGF)-induced tubule formation, as observed in an in vitro angiogenesis assays. In contrast with structurally different heparin, these cellulose sulfates have no apparent affinity to VEGF. Their impact on endothelial function may possibly be due to interactions with cell surface receptors/soluble factors not yet defined.

Biphasic Scaffolds from Marine Collagens for Regeneration of Osteochondral Defects.

BACKGROUND: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). METHODS: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-ß on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. RESULTS: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. CONCLUSIONS: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

Non-functionalized soft alginate hydrogel promotes locomotor recovery after spinal cord injury in a rat hemimyelonectomy model.

BACKGROUND: Spinal cord injury (SCI) and the consecutive devastating neurological sequelae have an enormous individual and economic impact. Implantation of functionalized hydrogels is a promising approach, because they can serve as a matrix for the regenerating tissue, carry and release bioactive molecules and various cell types. We already demonstrated that non-functionalized soft alginate hydrogel supported axonal outgrowth and protected neurons against oxidative stress in vitro. Here, we investigated the effects of such soft alginate hydrogels on locomotor recovery in small and large spinal cord lesions. METHOD: Hemimyelonectomy of 2 mm or 4 mm length was performed in rats and soft alginate hydrogel was implanted. Functional recovery of the hindlimbs was assessed in the open field [Batto Beattie Bresnahan (BBB) score] and using swimming test [Louisville Swim score (LSS)] for 140 days post injury (DPI). Reference histology was performed. RESULTS: Rats that received an alginate implant into 2 mm spinal cord lesions demonstrated significantly improved locomotor recovery compared to controls detectable already at 10 DPI. At 140 DPI, they reached higher LSS and BBB scores in swimming and open field tests, respectively. However, this beneficial effect of alginate was lacking in animals with larger (4 mm) lesions. Histological examination suggested that fibrous scarring in the spinal cord was reduced after alginate implantation in comparison to controls. CONCLUSIONS: Implantation of soft alginate hydrogel in small spinal cord lesions improved functional recovery. Possible underlying mechanisms include the mechanical stabilization of the wound, reduction of secondary damage and inhibition of fibrous scarring.

Novel alginate biphasic scaffold for osteochondral regeneration: an in vivo evaluation in rabbit and sheep models.

Current therapeutic strategies for osteochondral restoration showed a limited regenerative potential. In fact, to promote the growth of articular cartilage and subchondral bone is a real challenge, due to the different functional and anatomical properties. To this purpose, alginate is a promising biomaterial for a scaffold-based approach, claiming optimal biocompatibility and good chondrogenic potential. A previously developed mineralized alginate scaffold was investigated in terms of the ability to support osteochondral regeneration both in a large and medium size animal model. The results were evaluated macroscopically and by microtomography, histology, histomorphometry, and immunohistochemical analysis. No evidence of adverse or inflammatory reactions was observed in both models, but limited subchondral bone formation was present, together with a slow scaffold resorption time.The implantation of this biphasic alginate scaffold provided partial osteochondral regeneration in the animal model. Further studies are needed to evaluate possible improvement in terms of osteochondral tissue regeneration for this biomaterial.

Effects of a Pasty Bone Cement Containing Brain-Derived Neurotrophic Factor-Functionalized Mesoporous Bioactive Glass Particles on Metaphyseal Healing in a New Murine Osteoporotic Fracture Model.

The development of new and better implant materials adapted to osteoporotic bone is still urgently required. Therefore, osteoporotic muscarinic acetylcholine receptor M3 (M3 mAChR) knockout (KO) and corresponding wild type (WT) mice underwent osteotomy in the distal femoral metaphysis. Fracture gaps were filled with a pasty α-tricalcium phosphate (α-TCP)-based hydroxyapatite (HA)-forming bone cement containing mesoporous bioactive CaP-SiO2 glass particles (cement/MBG composite) with or without Brain-Derived Neurotrophic Factor (BDNF) and healing analyzed after 35 days. Histologically, bone formation was significantly increased in WT mice that received the BDNF-functionalized cement/MBG composite compared to control WT mice without BDNF. Cement/MBG composite without BDNF increased bone formation in M3 mAChR KO mice compared to equally treated WT mice. Mass spectrometric imaging showed that the BDNF-functionalized cement/MBG composite implanted in M3 mAChR KO mice was infiltrated by newly formed tissue. Leukocyte numbers were significantly lower in M3 mAChR KO mice treated with BDNF-functionalized cement/MBG composite compared to controls without BDNF. C-reactive protein (CRP) concentrations were significantly lower in M3 mAChR KO mice that received the cement/MBG composite without BDNF when compared to WT mice treated the same. Whereas alkaline phosphatase (ALP) concentrations in callus were significantly increased in M3 mAChR KO mice, ALP activity was significantly higher in WT mice. Due to a stronger effect of BDNF in non osteoporotic mice, higher BDNF concentrations might be needed for osteoporotic fracture healing. Nevertheless, the BDNF-functionalized cement/MBG composite promoted fracture healing in non osteoporotic bone.

Anti-skeletal muscle atrophy effect of Oenothera odorata root extract via reactive oxygen species-dependent signaling pathways in cellular and mouse model.

Skeletal muscle atrophy can be defined as a decrease of muscle volume caused by injury or lack of use. This condition is associated with reactive oxygen species (ROS), resulting in various muscular disorders. We acquired 2D and 3D images using micro-computed tomography in gastrocnemius and soleus muscles of sciatic-denervated mice. We confirmed that sciatic denervation-small animal model reduced muscle volume. However, the intraperitoneal injection of Oenothera odorata root extract (EVP) delayed muscle atrophy compared to a control group. We also investigated the mechanism of muscle atrophy's relationship with ROS. EVP suppressed expression of SOD1, and increased expression of HSP70, in both H2O2-treated C2C12 myoblasts and sciatic-denervated mice. Moreover, EVP regulated apoptotic signals, including caspase-3, Bax, Bcl-2, and ceramide. These results indicate that EVP has a positive effect on reducing the effect of ROS on muscle atrophy.

Materials and scaffolds in medical 3D printing and bioprinting in the context of bone regeneration.

The structural and functional repair of lost bone is still one of the biggest challenges in regenerative medicine. In many cases, autologous bone is used for the reconstruction of bone tissue; however, the availability of autologous material is limited, which always means additional stress to the patient. Due to this, more and more frequently various biocompatible materials are being used instead for bone augmentation. In this context, in order to ensure the structural function of the bone, scaffolds are implanted and fixed into the bone defect, depending on the medical indication. Nevertheless, for the surgeon, every individual clinical condition in which standardized scaffolds have to be aligned is challenging, and in many cases the alignment is not possible without limitations. Therefore, in the last decades, 3D printing (3DP) or additive manufacturing (AM) of scaffolds has become one of the most innovative approaches in surgery to individualize and improve the treatment of patients. Numerous biocompatible materials are available for 3DP, and various printing techniques can be applied, depending on the process conditions of these materials. Besides these conventional printing techniques, another promising approach in the context of medical AM is 3D bioprinting, a technique which makes it possible to print human cells embedded in special carrier substances to generate functional tissues. Even the direct printing into bone defects or lesions becomes possible. 3DP is already improving the treatment of patients, and has the potential to revolutionize regenerative medicine in future.

Hypoxia-conditioned media allows species-specific attraction of bone marrow stromal cells without need for recombinant proteins.

BACKGROUND: In vivo tissue regeneration depends on migration of stem cells into injured areas, their differentiation into specific cell types, and their interaction with other cells that are necessary to generate new tissue. Human mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs), can migrate and differentiate into osteoblasts in bone tissue. This can be facilitated by recombinant growth factors and cytokines. In many animal species, the availability of genomic sequences, recombinant proteins, and/or antibodies is limited so that new approaches are needed to generate resources that facilitate migration of stem cells into tissue defect areas. Here we used bone marrow stromal cells of human, ovine, equine, and canine origin to generate hypoxia-conditioned media (HCM) in order to attract BMSCs of the respective species in migration assays. RESULTS: We show that HCM contain attractors even more potent than vascular endothelial growth factor and can therefore be used in many animal species without the need for purified proteins. CONCLUSION: Generation of HCM is easy and cheap compared to preparation and purification of protein fractions and/or recombinant proteins. Hence, HCM could be applied in large animals (e.g. sheep, horse, dogs) for attraction of BMSCs into tissue defects caused by tumor resection or trauma.