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3D inkjet printing is a fast, reliable, and non‐contact bottom–up approach to printing small and large models and is one of the fastest additive manufacturing technologies available. These attributes position inkjet printing as a promising tool for the additive manufacturing of biomaterials, for example, tissue engineering scaffolds. However, due to the stringent technical rheological requirements of current inkjet technologies, there is a lack of photopolymer resins suitable for the inkjet printing of biomaterials. Hence, a novel ink engineered for 3D piezoelectric inkjet printing of biomaterials is designed and developed. The novel resin leverages a biodegradable amino acid phosphorodiamidate matrix copolymerized with a dialkyne ether to modulate the viscosity. Copolymerization with commercially available thiols facilitates the photochemical thiol‐yne curing reaction. The ink exhibits optimal viscosity, eliminating the need for solvents, as well as reliable jetting and sufficiently swift curing kinetics. Furthermore, the formulation is successfully demonstrated in an industrial inkjet printhead. Notably, the resulting materials have low cytotoxicity and, hence, have significant promise in advancing the applications of 3D inkjet printing of biological scaffolds. [ABSTRACT FROM AUTHOR]

Stereolithographic bioprinting holds great promise in the quest for creating artificial, biomimetic cartilage‐like tissue. To introduce a more biomimetic approach, we examined blending and stratifying methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) bioinks to mimic the zonal structure of articular cartilage. Bioinks were suspended with porcine chondrocytes before being printed in a digital light processing approach. Homogenous constructs made from hybrid bioinks of varying polymer ratios as well as stratified constructs combining different bioink blends were cultivated over 14 days and analyzed by histochemical staining for proteoglycans/collagen type II, cartilage marker expression analysis, and for cellular viability. The stiffness of blended bioinks increased gradually with HAMA content, from 2.41 ± 0.58 kPa (5% GelMA, 0% HAMA) to 8.84 ± 0.11 kPa (0% GelMA, 2% HAMA). Cell‐laden constructs maintained vital chondrocytes and supported the formation of proteoglycans and collagen type II. Higher concentrations of GelMA resulted in increased formation of cartilaginous matrix proteins and a more premature phenotype. However, decreased matrix production in central areas of constructs was observed in higher GelMA content constructs. Biomimetically stratified constructs retained their gradient‐like structure even after ECM formation, and exclusively exhibited a significant increase in COL2A1 gene expression (+178%). Concluding, we showed the feasibility of blending and stratifying photopolymerizable, natural biopolymers by SLA bioprinting to modulate chondrocyte attributes and to create zonally segmented ECM structures, contributing to improved modeling of cartilaginous tissue for regenerative therapies or in vitro models. [ABSTRACT FROM AUTHOR]

To create artificial cartilage in vitro, mimicking the function of native extracellular matrix (ECM) and morphological cartilage‐like shape is essential. The interplay of cell patterning and matrix concentration has high impact on the phenotype and viability of the printed cells. To advance the capabilities of cartilage bioprinting, we investigated different ECMs to create an in vitro chondrocyte niche. Therefore, we used methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) in a stereolithographic bioprinting approach. Both materials have been shown to support cartilage ECM formation and recovery of chondrocyte phenotype. We used these materials as bioinks to create cartilage models with varying chondrocyte densities. The models maintained shape, viability, and homogenous cell distribution over 14 days in culture. Chondrogenic differentiation was demonstrated by cartilage‐typical proteoglycan and type II collagen deposition and gene expression (COL2A1, ACAN) after 14 days of culture. The differentiation pattern was influenced by cell density. A high cell density print (25 × 106 cells/mL) led to enhanced cartilage‐typical zonal segmentation compared to cultures with lower cell density (5 × 106 cells/mL). Compared to HAMA, GelMA resulted in a higher expression of COL1A1, typical for a more premature chondrocyte phenotype. Both bioinks are feasible for printing in vitro cartilage with varying differentiation patterns and ECM organization depending on starting cell density and chosen bioink. The presented technique could find application in the creation of cartilage models and in the treatment of articular cartilage defects using autologous material and adjusting the bioprinted constructs size and shape to the patient. © 2019 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2649–2657, 2019. [ABSTRACT FROM AUTHOR]

There is evidence that the application of mesenchymal stromal cells (MSCs) counteracts osteoarthritis (OA) progression. However, the prospect of extracting and expanding these cells might be limited. The aim of this study was to investigate whether hyaluronic acid (HA) supplemented with MSC-recruiting chemokine C-C motif ligand 25 (CCL25) can influence the natural course of spontaneous OA in the guinea pig. CCL25 concentration in synovial fluid (SF) was quantified with enzyme-linked immunosorbent assay. Boyden chamber cell migration assay was used to test CCL25-mediated migration of guinea pig MSC. Forty-nine 11-month-old male guinea pigs were divided into seven groups. The main treatments consisted of five intra-articular injections of HA in pure form and in combination with three doses of CCL25 (63, 693, and 6,993 pg) given at a weekly interval. The severity of cartilage damage was assessed by using a modified Mankin score. The measured average physiological concentration of CCL25 in SF of animals is 85 ± 39 pg/ml. MSC showed a 3.2-fold increase in cell migration at 1,000 nM CCL25 in vitro demonstrating the biological migratory activity of CCL25 on these cells. In vivo, treatment with HA alone did not reduce OA progression. Similarly, OA scores were not found significantly reduced after treatment with 63 pg CCL25 +HA. However, when compared to pure HA, treatment with 693 pg CCL25 + HA and 6,993 pg CCL25 +HA significantly reduced the OA score from 10.1 to 7.4 (-28%) and 8.4 (-20%), respectively. These data suggest that intra-articular injections of HA supplemented with CCL25 attenuates OA. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the potential of an anti‐inflammatory polyethylene glycol (PEG) hydrogel for osteoarthritis (OA) management in an OA in vitro model. Freshly isolated porcine chondrocytes were maintained in high‐density cultures to form cartilage‐like three‐dimensional micromasses. Recombinant porcine tumor necrosis factor‐alpha (TNF‐α) was used to induce OA‐like changes. Normal and OA‐like micromasses were treated with dendritic polyglycerol sulfate‐based PEG hydrogel. Live/dead staining showed that all micromasses remained vital and presented similar morphological characteristics. Safranin‐O staining demonstrated a typical depletion of glycosaminoglycans in TNF‐α‐treated micromasses but not in the presence of the hydrogel. There was no distinct difference in immunohistochemical detection of type II collagen. Microarray data showed that rheumatoid arthritis and TNF signaling pathways were down regulated in hydrogel‐treated OA‐like micromasses compared to nontreated OA‐like micromasses. The hydrogel alone did not affect genes related to OA such as ANPEP, COMP, CXCL12, PTGS2, and TNFSF10, but it prevented their regulation caused by TNF‐α. This study provides valuable insights toward a fully synthetic hydrogel for the intra‐articular treatment of OA. The findings proved the potential of this hydrogel to prevent the development of TNF‐α‐induced OA with regard to proteoglycan loss and TNF‐α‐induced expression pattern without additional signs of differentiation and inflammation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 490–500, 2019. [ABSTRACT FROM AUTHOR]

Tendon healing is a time consuming process leading to the formation of a functionally altered reparative tissue. Tissue engineering-based tendon reconstruction is attracting more and more interest. The aim of this study was to establish tenocyte expansion on microcarriers in continuous bioreactor cultures and to study tenocyte behavior during this new approach. Human hamstring tendon-derived tenocytes were expanded in monolayer culture before being seeded at two different seeding densities (2.00 and 4.00 × 106 cells/1000 cm2 surface) on Cytodex™ type 3 microcarriers. Tenocytes' vitality, growth kinetics and glucose/lactic acid metabolism were determined dependent on the seeding densities and stirring velocities (20 or 40 rpm) in a spinner flask bioreactor over a period of 2 weeks. Gene expression profiles of tendon extracellular matrix (ECM) markers (type I/III collagen, decorin, cartilage oligomeric protein [COMP], aggrecan) and the tendon marker scleraxis were analyzed using real time detection polymerase chain reaction (RTD-PCR). Type I collagen and decorin deposition was demonstrated applying immunolabeling. Tenocytes adhered on the carriers, remained vital, proliferated and revealed an increasing glucose consumption and lactic acid formation under all culture conditions. 'Bead-to-bead' transfer of cells from one microcarrier to another, a prerequisite for continuous tenocyte expansion, was demonstrated by scanning electron microscopy. Type I and type III collagen gene expression was mainly unaffected, whereas aggrecan and partly also decorin and COMP expression was significantly downregulated compared to monolayer cultures. Scleraxis gene expression revealed no significant regulation on the carriers. In conclusion, tenocytes could be successfully expanded on microcarriers. Therefore, bioreactors are promising tools for continuous tenocyte expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:142-151, 2014 [ABSTRACT FROM AUTHOR]

Chitosan-beta glycerophosphate-hydroxyethyl cellulose (CH-GP-HEC) is a biocompatible and biodegradable scaffold exhibiting a sol-gel transition at 37°C. Chondrogenic factors or mesenchymal stem cells (MSCs) can be included in the CH-GP-HEC, and injected into the site of injury to fill the cartilage tissue defects with minimal invasion and pain. The possible impact of the injectable CH-GP-HEC on the viability of the encapsulated MSCs was assessed by propidium iodide-fluorescein diacetate staining. Proliferation of the human and rat MSCs was also determined by MTS assay on days 0, 7, 14 and 28 after encapsulation. To investigate the potential application of CH-GP-HEC as a drug delivery device, the in vitro release profile of insulin was quantified by QuantiPro-BCA™ protein assay. Chondrogenic differentiation capacity of the encapsulated human MSCs (hMSCs) was also determined after induction of differentiation with transforming growth factor β3. MSCs have very good survival and proliferative rates within CH-GP-HEC hydrogel during the 28-day investigation. A sustained release of insulin occurred over 8 days. The CH-GP-HEC hydrogel also provided suitable conditions for chondrogenic differentiation of the encapsulated hMSCs. In conclusion, the high potential of CH-GP-HEC as an injectable hydrogel for cartilage tissue engineering is emphasised. [ABSTRACT FROM AUTHOR]

Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell-cell and cell-matrix contacts within three-dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic-co-glycolic-acid] (PLGA)-scaffolds and high-density cultures. Neotissue formation was examined by hematoxyline-eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon-like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4-week-old 3D cultures, whereas type III collagen was only elevated in high-density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high-density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon-like tissue. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1170-1177, 2010 [ABSTRACT FROM AUTHOR]

The increasing prevalence of cartilage destruction during arthritis has entailed an intensified amount for in vitro cartilage models to analyze pathophysiological processes and to screen for antirheumatic drugs. Tissue engineering offers the opportunity to establish highly organized 3D cell cultures facilitating the formation of in vitro models that reflect the human situation. We report the comparison of porcine chondrocyte pellet and alginate bead cultures as model systems for human cartilage and the further development into a human system that was applied in an arthritis model. In porcine pellet and alginate cultures, formation of cartilage matrix similar to human matrix was verified by histology and PCR. As alginate beads could be cultivated batch-wise in one well of a multiwell plate, we further developed this setting into a human system. In contrast, each pellet had to be cultivated individually in one well of a multiwell plate, which is time consuming. Following stimulation of human chondrocyte alginate cultures with conditioned media from human synovial fibroblasts derived from arthritis patients, microarray analysis verified the induction of genes related to cartilage destruction (like MMP10, -12) and inflammation (like IL6, -8 and chemokines). Several genes are coding for proteins that are members of inflammatory and catabolic pathways. Belonging to the most affected pathways, we identified the focal adhesion, cytokine–cytokine receptor interaction, ECM-receptor signalling, Jak-STAT signalling, and toll-like receptor signalling pathways, all relevant in arthritis. Therefore, we demonstrate that engineered cartilage of porcine and human origin represents a powerful in vitro model for cartilage in vivo. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the osteogenic effect of three different cell‐seeded 3D‐bioplotted scaffolds in a ovine calvarial critical‐size defect model. The choice of scaffold‐materials was based on their applicability for 3D‐bioplotting and respective possibility to produce tailor‐made scaffolds for the use in cranio‐facial surgery for the replacement of complex shaped boneparts. Scaffold raw‐materials are known to be osteoinductive when being cell‐seeded [poly(L‐lactide‐co‐glycolide) (PLGA)] or having components with osteoinductive properties as tricalciumphosphate (TCP) or collagen (Col) or chitosan. The scaffold‐materials PLGA, TCP/Col, and HYDR (TCP/Col/chitosan) were cell‐seeded with osteoblast‐like cells whether gained from bone (OLB) or from periost (OLP). In a prospective and randomized design nine sheep underwent osteotomy to create four critical‐sized calvarial defects. Three animals each were assigned to the HYDR‐, the TCP/Col‐, or the PLGA‐group. In each animal, one defect was treated with a cell‐free, an OLB‐ or OLP‐seeded group‐specific scaffold, respectively. The fourth defect remained untreated as control (UD). Fourteen weeks later, animals were euthanized for histo‐morphometrical analysis of the defect healing. OLB‐ and OLP‐seeded HYDR and OLB‐seeded TCP/Col scaffolds significantly increased the amount of newly formed bone (NFB) at the defect bottom and OLP‐seeded HYDR also within the scaffold area, whereas PLGA‐scaffolds showed lower rates. The relative density of NFB was markedly higher in the HYDR/OLB group compared to the corresponding PLGA group. TCP/Col had good stiffness to prepare complex structures by bioplotting but HYDR and PLGA were very soft. HYDR showed appropriate biodegradation, TCP/Col and PLGA seemed to be nearly undegraded after 14 weeks. 3D‐bioplotted, cell‐seeded HYDR and TCP/Col scaffolds increased the amount of NFB within ovine critical‐size calvarial defects, but stiffness, respectively, biodegradation of materials is not appropriate for the application in cranio‐facial surgery and have to be improved further by modifications of the manufacturing process or their material composition. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010 [ABSTRACT FROM AUTHOR]

The aim of our study was to evaluate the mid-term outcome of a cell-free polymer-based cartilage repair approach in a sheep cartilage defect model in comparison to microfracture treatment. Cell-free, freeze-dried implants (chondrotissue®) made of a poly-glycolic acid (PGA) scaffold and hyaluronan were immersed in autologous serum and used for covering microfractured full-thickness articular cartilage defects of the sheep ( n = 4). Defects treated with microfracture only served as controls ( n = 4). Six months after implantation, cartilage implants and controls were analyzed by immunohistochemical staining of type II collagen, histological staining of proteoglycans, and histological scoring. Histological analysis showed the formation of a cartilaginous repair tissue rich in proteoglycans. Histological scoring documented significant improvement of repair tissue formation when the defects were covered with the cell-free implant, compared to controls treated with microfracture. Immunohistochemistry showed that the cell-free implant induced cartilaginous repair tissue and type II collagen. Controls treated with microfracture showed marginal formation of a mixed-type repair tissue consisting of cartilaginous tissue and fibro-cartilage. Covering of microfractured defects with the cell-free polymer-based cartilage implant is suggested to be a promising treatment option for cartilage defects and improves the regeneration of articular cartilage. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1353-1360, 2009 [ABSTRACT FROM AUTHOR]

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Microfracture is a frequently used reparative technique that induces a healing response in articular cartilage defects. Penetration of the subchondral bone leads to blood clot formation, allows multipotent mesenchymal cells to access the defect and, subsequently, leads to cartilaginous repair tissue. The aim of our study was to analyze the chemotactic recruitment of human subchondral spongious bone marrow-derived cells by synovial fluid (SF) from normal donors (ND), patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Subchondral spongious bone marrow-derived mesenchymal progenitors were isolated from bone cylinders after high tibial osteotomy and analyzed for the presence of stem cell-related cell surface antigens by flowcytometry. Recruitment of subchondral progenitors by normal SF and SF from donors with degenerated joint diseases was documented by using a modified Boyden chamber chemotaxis assay. The chemotaxis assay demonstrated that synovial fluid has the potential to recruit mesenchymal progenitors in vitro. SF from normal donors and patients with OA showed no difference in the potential to stimulate cell migration. SF obtained from RA donors showed significantly reduced cell recruitment compared to SF derived from OA patients ( p = 0.0054) and normal donors ( p < 0.0001). The chemotactic activity of SF obtained from normal donors and from patients with degenerative joint diseases suggests that SF may be actively involved in the migration of progenitors in cartilage defects after microfracture. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1299-1307, 2007 [ABSTRACT FROM AUTHOR]

Full‐thickness defects in articular cartilage can be functionally restored by autologous chondrocyte implantation (ACI). In past years, numerous types of scaffolds for tissue‐engineered cartilage implants have been developed and thoroughly characterized. However, the fixation stability of the implants has been rarely investigated despite its well‐known importance for successful therapy. In this study, we have mechanically tested the fixation stability of four commonly used biomaterials for ACI attached by four different fixation techniques (unfixed, fibrin glue, chondral suture, and transosseous suture) in situ. Scaffolds based on polyglycolic acid (PGA) and polyglycolic acid and poly‐L‐lactic acid (PGLA), collagen membranes, and a gel‐like matrix material were fixed within rectangular full‐thickness cartilage defects of 10 × 15 mm2 and loaded in tension until failure. Fibrin glue fixation of PGLA‐scaffolds withstood a load of 2.18 6 ± 0.47 N, chondral sutured PGA‐scaffolds of 26.29 6 ± 1.55 N, and transosseous fixed PGA‐scaffolds of 38.18 6 ± 9.53 N. The PGA‐scaffold could be loaded highest until failure for all fixation techniques compared to the PGLA‐scaffold and collagen membrane. Our findings serve as basis for selecting the most suitable fixation technique for scaffold‐based tissue‐engineered grafts according to the expected in vivo loads. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007 [ABSTRACT FROM AUTHOR]

The functional roles of BMP type IA and IB receptors mediating differentiation into the osteogenic and chondrogenic lineage were investigated in the mesenchymal progenitor line C3H10T1/2 in vitro. The capacity of type IA and IB BMP receptors was assessed by the forced expression of the wild-type (wtBMPR-IA or IB) and of the kinase-deficient, dominant-negative form (dnBMPR-IA or -IB) in parental C3H10T1/2 progenitors as well as in C3H10T1/2 progenitors which recombinantly express BMP2 (C3H10T1/2-BMP2) or GDF5 (C3H10T1/2-GDF5). Consistent with the higher endogenous expression rate of BMPR-IA in comparison with BMPR-IB, BMPR-IA plays the dominant role in BMP2-mediated osteo-/chondrogenic development. BMPR-IB moderately influences osteogenic and hardly chondrogenic development. BMPR-IB seems to be unable to efficiently activate downstream signaling pathways upon forced expression. However, a mutation conferring constitutive activity to the BMPR-IB receptor indicates that this receptor possesses the capacity to activate downstream signaling cascades. These results suggest that in mesenchymal progenitors C3H10T1/2 BMPR-IA is responsible for the initiation of the osteogenic as well as chondrogenic development and that BMPR-IA and -IB receptor pathways are well separated in this mesenchymal progenitor line and may not substitute each other. In addition this indicates that type IB and IA BMP receptors may transmit different signals during the specification and differentiation of mesenchymal lineages. [ABSTRACT FROM AUTHOR]

Objective: For optimized expansion of human‐induced pluripotent stem cells (hiPSCs) with regards to clinical applications, we investigated the influence of the inoculum density on the expansion procedure in 3D hollow‐fibre bioreactors. Materials and Methods: Analytical‐scale bioreactors with a cell compartment volume of 3 mL or a large‐scale bioreactor with a cell compartment volume of 17 mL were used and inoculated with either 10 × 106 or 50 × 106 hiPSCs. Cells were cultured in bioreactors over 15 days; daily measurements of biochemical parameters were performed. At the end of the experiment, the CellTiter‐Blue® Assay was used for culture activity evaluation and cell quantification. Also, cell compartment sections were removed for gene expression and immunohistochemistry analysis. Results: The results revealed significantly higher values for cell metabolism, cell activity and cell yields when using the higher inoculation number, but also a more distinct differentiation. As large inoculation numbers require cost and time‐extensive pre‐expansion, low inoculation numbers may be used preferably for long‐term expansion of hiPSCs. Expansion of hiPSCs in the large‐scale bioreactor led to a successful production of 5.4 × 109 hiPSCs, thereby achieving sufficient cell amounts for clinical applications. Conclusions: In conclusion, the results show a significant effect of the inoculum density on cell expansion, differentiation and production of hiPSCs, emphasizing the importance of the inoculum density for downstream applications of hiPSCs. Furthermore, the bioreactor technology was successfully applied for controlled and scalable production of hiPSCs for clinical use. [ABSTRACT FROM AUTHOR]

The authors of the article titled "Fixation Stability of Scaffold-Based Tissue Engineered Grafts: Mechanical Testing of Fixation Techniques for Scaffold-Based Tissue Engineered Grafts" respond to Dr. Sven Kili's comments on their study. They clarify that their study aimed to measure the primary fixation strength of commonly used biomaterials for autologous chondrocyte implantation (ACI), rather than perform product testing on various scaffold materials. They acknowledge the limitations of their testing approach and the possibility of weakening during fixation. They conclude by presenting a procedure to test the postoperative stability of biomaterials for ACI. [Extracted from the article]

Microcarrier cultures have been shown to allow extensive cell expansion of tissue engineering relevant cells, such as chondrocytes, while maintaining their phenotype. Our aim was to investigate the in vitro three-dimensional expansion of porcine bone-marrow-derived primary mesenchymal stem cells (MSC) using commercially available Cytodex type 1, type 2, and type 3 microcarriers. In comparison, the Cytodex type 1 microcarriers showed the best results for adherence with over 80% adherent cells after 3 h of incubation, analyzed by the Poisson distribution. Different start cell densities ranging from 1 to 3 x 106 cells per 100 cm2 had only a minor influence on adhesion. The proliferation was examined on Cytodex type 1 microcarriers over a cultivation time of 28 days, which could reveal cell growth and proof of cells recolonizing freshly added microcarriers. Scanning electron microscopy displayed appropriate cell morphology and confirmed cell proliferation. After enzymatic harvest from microcarriers, the osteogenic and chondrogenic differentiation of these cells was induced and shown by relevant histochemistry, such as von Kossa and Alcian blue staining. Totaling the results, we have shown that the three-dimensional expansion of MSC on microcarriers represents a beneficial alternative to the conventional two-dimensional monolayer cultivation method.