Inflammatory profiles in canine intervertebral disc degeneration.

BACKGROUND: Intervertebral disc (IVD) disease is a common spinal disorder in dogs and degeneration and inflammation are significant components of the pathological cascade. Only limited studies have studied the cytokine and chemokine profiles in IVD degeneration in dogs, and mainly focused on gene expression. A better understanding is needed in order to develop biological therapies that address both pain and degeneration in IVD disease. Therefore, in this study, we determined the levels of prostaglandin E2 (PGE2), cytokines, chemokines, and matrix components in IVDs from chondrodystrophic (CD) and non-chondrodystrophic (NCD) dogs with and without clinical signs of IVD disease, and correlated these to degeneration grade (according to Pfirrmann), or herniation type (according to Hansen). In addition, we investigated cyclooxygenase 2 (COX-2) expression and signs of inflammation in histological IVD samples of CD and NCD dogs. RESULTS: PGE2 levels were significantly higher in the nucleus pulposus (NP) of degenerated IVDs compared with non-degenerated IVDs, and in herniated IVDs from NCD dogs compared with non-herniated IVDs of NCD dogs. COX-2 expression in the NP and annulus fibrosus (AF), and proliferation of fibroblasts and numbers of macrophages in the AF significantly increased with increased degeneration grade. GAG content did not significantly change with degeneration grade or herniation type. Cytokines interleukin (IL)-2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, immune protein (IP)-10, tumor necrosis factor (TNF)-α, and granulocyte macrophage colony-stimulating factor (GM-CSF) were not detectable in the samples. Chemokine (C-C) motif ligand (CCL)2 levels in the NP from extruded samples were significantly higher compared with the AF of these samples and the NP from protrusion samples. CONCLUSIONS: PGE2 levels and CCL2 levels in degenerated and herniated IVDs were significantly higher compared with non-degenerated and non-herniated IVDs. COX-2 expression in the NP and AF and reactive changes in the AF increased with advancing degeneration stages. Although macrophages invaded the AF as degeneration progressed, the production of inflammatory mediators seemed most pronounced in degenerated NP tissue. Future studies are needed to investigate if inhibition of PGE2 levels in degenerated IVDs provides effective analgesia and exerts a protective role in the process of IVD degeneration and the development of IVD disease.

The Injection of Gels Through an Intact Annulus Maintains Biomechanical Performance without Extrusion Risk.

For autologous-disc-derived chondrocyte transplantation (ADCT) a transglutaminase crosslinked gelatine gel and an albumin hyaluronic acid gel, crosslinked with bis-thio-polyethylene glycol, were injected through a syringe into a degenerated intervertebral disc, where they solidified in situ. This biomechanical in vitro study with lumbar bovine motion segments evaluated disc height changes, motion characteristics in a quasi-static spine loading simulators, and the potential extrusion risk of these biomaterials in a complex dynamic multi-axial loading set-up with 100,000 loading cycles. After the injection and formation of the gel in the center of the nucleus, the disc height increase was about 0.3 mm. During cyclic testing, a gradual decrease in height could be detected due to viscoelastic effects and fluid loss. No gel extrusion could be observed for all specimens during the entire test procedure. A macroscopic inspection after dissections showed an accumulation of the solidified gel in the center of the nucleus. The results demonstrate that the injection of in situ solidifying gels through the intact annulus allows for the stable maintenance of the injected gel at the target location, with high potential for use as a suitable scaffold to anchor therapeutically applied cells for disc regeneration within the treated nucleus pulposus.

Maintenance of "stem cell" features of cartilage cell sub-populations during in vitro propagation.

BACKGROUND: The discovery of mesenchymal stem cells (MSCs) or MSC-like cells in cartilage tissue does not tie in well with the established view that MSCs derive from a perivascular niche. The presence of MSCs may raise concerns about specificity and application safety, particularly in terms of the regulatory site. The aim of the present study was to investigate the benefits or possible risks of the MSC-like properties of cells isolated from cartilage in the context of autologous chondrocyte implantation. METHODS: Chondrocytic cells were isolated from cartilage or intervertebral disc tissue. Flow cytometry was used to analyze the expression of cell surface antigens. MSC-like cells were either enriched or depleted by means of magnetic cell sorting (MACS) involving the monoclonal antibodies W5C5/SUSD2 and W8B2/MSCA-1. We addressed the issues of prolonged expansion of such cells as well as the influence of culture medium as a trigger for selecting a single cell type. Established protocols were used to study in vitro differentiation. In addition to histological and biochemical assessment, the acquired phenotypes were also evaluated on the mRNA transcript level. RESULTS: In the studied cells, we found strongly analogous expression of antigens typically expressed on MSCs, including CD49e, CD73, CD90, CD105, CD140b and CD166. The expression of W5C5 and W8B2 antigens in cartilage cell sub-populations did not correlate with multi-potency. We demonstrated that a chondroid precursor, but not a bona fide multipotent mesenchymal, cell type can be obtained under established in vitro culture conditions. The culture media used for expansion influenced the cell phenotype. CONCLUSIONS: The risk of adverse adipose or osseous differentiation is not posed by expanded chondrocyte cultures, even after enrichment of putative MSC-like cell populations by MACS. It is possible that this limited "stemness" in chondrocytes, expanded for use in ACI, may instead be beneficial as it allows re-differentiation under appropriate conditions despite prolonged times in culture.

Prognostic value of stress-gated 99m-technetium SPECT myocardial perfusion imaging: risk stratification of patients with multivessel coronary artery disease and prior coronary revascularization.

BACKGROUND: This study assessed the prognostic value of stress-gated 99mTc-sestamibi myocardial perfusion SPECT (MPS) in patients with multivessel coronary artery disease (CAD) and prior revascularization according to the presence and severity of ischemia. METHODS AND RESULTS: We studied the outcome of 472 patients with multivessel CAD and prior revascularization (coronary angioplasty, 290 patients; bypass surgery, 182 patients), who underwent exercise or dipyridamole 99mTc-sestamibi MPS for evaluation of ischemia. Visual scoring of perfusion images used 20 segments and a 5-point scale. Gated post-stress EF was automatically calculated. Endpoints included hard events: cardiac death (CD) and nonfatal myocardial infarction (MI). During a mean follow-up of 3.0 ± 1.0 years, 37 hard events occurred, including CD in 15 (3%) and MI in 22 (5%) patients. In a risk-adjusted multivariable Cox model, a history of prior MI, diabetes, abnormal MPS, moderate-to-severe ischemia, and post-stress EF <35% were important predictors of cardiac events. Four-year risk-adjusted survival was 97.9% for normal MPS, 87.3% for abnormal MPS with ischemia, and 82.1% for moderate-to-severe ischemia. CONCLUSIONS: Among patients with previous coronary revascularization, stress-gated 99mTc-sestamibi MPS provides prognostic information for the prediction of cardiac events. A normal perfusion scan confers an excellent prognosis and an exceedingly low hard event rate (<1%/year). The presence of moderate-to-severe ischemia or a post-stress EF <35% identifies patients at highest risk of subsequent cardiac events.

In Vitro Comparison of 2 Clinically Applied Biomaterials for Autologous Chondrocyte Implantation: Injectable Hydrogel Versus Collagen Scaffold.

OBJECTIVE: In autologous chondrocyte implantation (ACI), there is no consensus about used bioscaffolds. The aim of this study was to perform an in vitro comparative analysis of 2 clinically applied biomaterials for cartilage lesion treatment. DESIGN: Monolayer expanded human chondrocytes (n = 6) were embedded in a collagen scaffold (CS) and a hyaluronic acid-based hydrogel (HA). Cells were cultured in chondropermissive medium supplemented with and without interleukin-10 (IL-10) and bone morphogenetic protein-2 (BMP-2). Gene expression of chondrogenic markers (COL1A1, COL2A1, COL10A1, ACAN, SOX9) was detected via quantitative real-time-polymerase chain reaction (RT-qPCR). Biosynthesis of matrix compounds, cell viability, morphology as well as migration from surrounding native bovine cartilage into cell-free scaffolds were analyzed histologically. Adhesion of the material to adjacent cartilage was investigated by a custom-made push-out test. RESULTS: The shift of COL1/2 ratio toward COL2A1 was more pronounced in HA, and cells displayed a more spherical morphology compared with CS. BMP-2 and IL-10 significantly increased COL2A1, SOX9, and ACAN expression, which was paralleled by enhanced staining of glycosaminoglycans (GAGs) and type 2 collagen in histological sections of CS and HA. COL10A1 was not significantly expressed in HA and CS. Better interfacial integration and enhanced cell invasion was observed in CS. Push-out tests using CS showed higher bonding strength to native cartilage. CONCLUSION: HA-based hydrogel revealed a more chondrocyte-like phenotype but only allowed limited cell invasion, whereas CS were advantageous in terms of cellular invasion and interfacial adhesion. These differences may be clinically relevant when treating cartilaginous or osteochondral defects.

Intervertebral disc cell- and hydrogel-supported and spontaneous intervertebral disc repair in nucleotomized sheep.

PURPOSE: Regenerative repair is a promising new approach in treating damaged intervertebral discs. An experimental scheme was established for autologous and/or allogenic repair after massive disc injury. METHODS: Disc healing was promoted in 11 animals by injecting in vitro expanded autologous/homologous disc cells 2 weeks after stab injury of lumbar discs L1-2. The following control discs were used in our sheep injury model: L2-3, vehicle only; L3-4, injury only; L4-5, undamaged; and lumbar discs from four non-experimental animals. Disc cells were suspended in a biologically supportive albumin/hyaluronan two-component hydrogel solution that polymerizes when inserted in order to anchor cells at the injection site. The parameters studied were MRI, DNA, glycosaminoglycan, collagen content, histology, immunohistology for collagens type I, II and aggrecan, and mRNA expression of GAPDH, ß-actin, collagen type I, II, X, aggrecan, lubricin, and IL-1ß. RESULTS: All parameters demonstrated almost complete healing of the injured discs after 6 months, when compared with data from both the endogenous non-injured controls as well as from the healthy animals. CONCLUSION: Sheep experience spontaneous recovery from disc injury. The process of endogenous repair can be enhanced by means of hydrogel-supported cells.

Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair.

BACKGROUND: Cell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue. METHODS: A hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured in vitro and in vivo in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific in situ hybridization was performed to discriminate between cells of human and murine origin in xenotransplants. RESULTS: The reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. In vitro and in vivo (subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels in vitro and in vivo, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the in vitro cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific in situ hybridization. CONCLUSIONS: The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.

"Artificial micro organs"--a microfluidic device for dielectrophoretic assembly of liver sinusoids.

In order to study possible toxic side effects of potential drug compounds in vitro a reliable test system is needed. Predicting liver toxicity presents a major challenge of particular importance as liver cells grown in a cell culture suffer from a rapid loss of their liver specific functions. Therefore we are developing a new microfluidic test system for liver toxicity. This test system is based on an organ-like liver 3D co-culture of hepatocytes and endothelial cells. We devised a microfluidic chip featuring cell culture chambers with integrated electrodes for the assembly of liver sinusoids by dielectrophoresis. Fluid channels enable an organ-like perfusion with culture media and test compounds. Different chamber designs were studied and optimized with regard to dielectrophoretic force distribution, hydrodynamic flow profile, and cell trapping rate using numeric simulations. Based on simulation results a microchip was injection-moulded from COP. This chip allowed the assembly of viable hepatocytes and endothelial cells in a sinusoid-like fashion.

A method for patterned in situ biofunctionalization in injection-molded microfluidic devices.

We developed a method to modify the surface in injection molded polymer microdevices prior to bonding and to pattern biomolecules in the completed microsystem in situ by a sequence of simple perfusion steps directly before utilization of the device. This method is compatible with production technology such as injection molding and bonding processes currently employed in the fabrication of polymer microsystems. It solves the problem of the inherent incompatibility of biomolecules with microfabrication technology as it allows for the biofunctionalization step to be performed after completion of the microsystem. Injection molded cyclic olefin copolymer (COC) microfluidic chips were modified by irradiating the surface with UV-light at lambda = 185 nm. This results in the formation of stable acidic groups which were further modified by binding of the extracellular matrix protein collagen type I. Non-irradiated surfaces were modified by binding of Pluronic® F-127 to become non-adhesive. Density of acid groups decreases to 50% within 45 days and to 25% within 19 weeks after irradiation. However, even then the remaining density of functional groups was shown to be sufficient to bind proteins and promote cell adhesion. Selective adhesion of primary hepatocytes on surfaces patterned by UV-irradiation and a biofunctional coating with collagen type I were demonstrated in injection molded microsystems.

Chondrogenic potential of human adult mesenchymal stem cells is independent of age or osteoarthritis etiology.

Osteoarthritis (OA) is a multifactorial disease strongly correlated with history of joint trauma, joint dysplasia, and advanced age. Mesenchymal stem cells (MSCs) are promising cells for biological cartilage regeneration. Conflicting data have been published concerning the availability of MSCs from the iliac crest, depending on age and overall physical fitness. Here, we analyzed whether the availability and chondrogenic differentiation capacity of MSCs isolated from the femoral shaft as an alternative source is age- or OA etiology-dependent. MSCs were isolated from the bone marrow (BM) of 98 patients, categorized into three OA-etiology groups (age-related, joint trauma, joint dysplasia) at the time of total hip replacement. All BM samples were characterized for cell yield, proliferation capacity, and phenotype. Chondrogenic differentiation was studied using micromass culture and analyzed by histology, immunohistochemistry, and quantitative reverse transcriptase-polymerase chain reaction. Significant volumes of viable BM (up to 25 ml) could be harvested from the femoral shaft without observing donor-site morbidity, typically containing >10(7) mononuclear cells per milliliter. No correlation of age or OA etiology with the number of mononuclear cells in BM, MSC yield, or cell size was found. Proliferative capacity and cellular spectrum of the harvested cells were independent of age and cause of OA. From all tested donors, MSCs could be differentiated into the chondrogenic lineage. We conclude that, irrespective of age and OA etiology, sufficient numbers of MSCs can be isolated and that these cells possess an adequate chondrogenic differentiation potential. Therefore, a therapeutic application of MSCs for cartilage regeneration of OA lesions seems feasible. Disclosure of potential conflicts of interest is found at the end of this article.

Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside.

The socioeconomic burden of chronic back pain related to intervertebral disc (IVD) disease is high and current treatments are only symptomatic. Minimally invasive strategies that promote biological IVD repair should address this unmet need. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) during IVD maturation and degeneration. The regenerative potential of NC-secreted substances on CLCs and mesenchymal stromal cells (MSCs) has already been demonstrated. However, identification of these substances remains elusive. Innovatively, this study exploits the regenerative NC potential by using healthy porcine NC-derived matrix (NCM) and employs the dog as a clinically relevant translational model. NCM increased the glycosaminoglycan and DNA content of human and canine CLC aggregates and facilitated chondrogenic differentiation of canine MSCs in vitro. Based on these results, NCM, MSCs and NCM+MSCs were injected in mildly (spontaneously) and moderately (induced) degenerated canine IVDs in vivo and, after six months of treatment, were analyzed. NCM injected in moderately (induced) degenerated canine IVDs exerted beneficial effects at the macroscopic and MRI level, induced collagen type II-rich extracellular matrix production, improved the disc height, and ameliorated local inflammation. MSCs exerted no (additive) effects. In conclusion, NCM induced in vivo regenerative effects on degenerated canine IVDs. NCM may, comparable to demineralized bone matrix in bone regeneration, serve as 'instructive matrix', by locally releasing growth factors and facilitating tissue repair. Therefore, intradiscal NCM injection could be a promising regenerative treatment for IVD disease, circumventing the cumbersome identification of bioactive NC-secreted substances.

Bone Morphogenetic Protein-2, But Not Mesenchymal Stromal Cells, Exert Regenerative Effects on Canine and Human Nucleus Pulposus Cells.

Chronic back pain is related to intervertebral disc (IVD) degeneration and dogs are employed as animal models to develop growth factor- and cell-based regenerative treatments. In this respect, the differential effects of transforming growth factor beta-1 (TGF-ß1) and bone morphogenetic protein-2 (BMP2) on canine and human chondrocyte-like cells (CLCs) derived from the nucleus pulposus of degenerated IVDs were studied. Human and canine CLCs were cultured in 3D microaggregates in basal culture medium supplemented with/without TGF-ß1 (10 ng/mL) or BMP2 (100 or 250 ng/mL). Both TGF-ß1 and BMP2 increased proliferation and glycosaminoglycan (GAG) deposition of human and canine CLCs. TGF-ß1 induced collagen type I deposition and fibrotic (re)differentiation, whereas BMP2 induced more collagen type II deposition. In dogs, TGF-ß1 induced Smad1 and Smad2 signaling, whereas in humans, it only tended to induce Smad2 signaling. BMP2 supplementation increased Smad1 signaling in both species. This altogether indicates that Smad1 signaling was associated with collagen type II production, whereas Smad2 signaling was associated with fibrotic CLC (re)differentiation. As a step toward preclinical translation, treatment with BMP2 alone and combined with mesenchymal stromal cells (MSCs) was further investigated. Canine male CLCs were seeded in albumin-based hydrogels with/without female bone marrow-derived MSCs (50:50) in basal or 250 ng/mL BMP2-supplemented culture medium. Although the results indicate that a sufficient amount of MSCs survived the culture period, total GAG production was not increased and GAG production per cell was even decreased by the addition of MSCs, implying that MSCs did not exert additive regenerative effects on the CLCs.

A Degenerative/Proinflammatory Intervertebral Disc Organ Culture: An Ex Vivo Model for Anti-inflammatory Drug and Cell Therapy.

Resolution of intervertebral disc (IVD) degeneration-associated inflammation is a prerequisite for tissue regeneration and could possibly be achieved by strategies ranging from pharmacological to cell-based therapies. In this study, a proinflammatory disc organ culture model was established. Bovine caudal disc punches were needle punctured and additionally stimulated with lipopolysaccharide (10 µg/mL) or interleukin-1ß (IL-1ß, 10-100 ng/mL) for 48 h. Two intradiscal therapeutic approaches were tested: (i) a nonsteroidal anti-inflammatory drug, diclofenac (Df) and (ii) human mesenchymal stem/stromal cells (MSCs) embedded in an albumin/hyaluronan hydrogel. IL-1ß-treated disc organ cultures showed a statistically significant upregulation of proinflammatory markers (IL-6, IL-8, prostaglandin E2 [PGE2]) and metalloproteases (MMP1, MMP3) expression, while extracellular matrix (ECM) proteins (collagen II, aggrecan) were significantly downregulated. The injection of the anti-inflammatory drug, Df, was able to reduce the levels of proinflammatory cytokines and MMPs and surprisingly increase ECM protein levels. These results point the intradiscal application of anti-inflammatory drugs as promising therapeutics for disc degeneration. In parallel, the immunomodulatory role of MSCs on this model was also evaluated. Although a slight downregulation of IL-6 and IL-8 expression could be found, the variability among the five donors tested was high, suggesting that the beneficial effect of these cells on disc degeneration needs to be further evaluated. The proinflammatory/degenerative IVD organ culture model established can be considered a suitable approach for testing novel therapeutic drugs, thus reducing the number of animals in in vivo experimentation. Moreover, this model can be used to address the cellular and molecular mechanisms that regulate inflammation in the IVD and their implications in tissue degeneration.

Human Placenta-Derived CD146-Positive Mesenchymal Stromal Cells Display a Distinct Osteogenic Differentiation Potential.

Mesenchymal stromal cells (MSCs) are multipotent cells that can be differentiated in vitro into a variety of cell types, including adipocytes or osteoblasts. Our recent studies indicated that a high expression of CD146 on MSCs from bone marrow correlates with their robust osteogenic differentiation potential. We therefore investigated if expression of CD146 on MSCs from the placenta correlates with a similar osteogenic differentiation potential. The MSCs were isolated specifically from the endometrial and fetal parts of human term placenta and expanded in separate cultures and compared with MSCs from bone marrow as controls. The expression of cell surface antigens was investigated by flow cytometry. Differentiation of MSCs was documented by cytochemistry and analysis of typical lineage marker genes. CD146-positive MSCs were separated from CD146-negative cells by magnet-assisted cell sorts (MACS). We report that the expression of CD146 is associated with a higher osteogenic differentiation potential in human placenta-derived MSCs (pMSCs) and the CD146(pos) pMSCs generated a mineralized extracellular matrix, whereas the CD146(neg) pMSCs failed to do so. In contrast, adipogenic and chondrogenic differentiation of pMSCs was not different in CD146(pos) compared with CD146(neg) pMSCs. Upon enrichment of pMSCs by MACS, the CD146(neg) and CD146(pos) populations maintained their expression levels for this antigen for several passages in vitro. We conclude that CD146(pos) pMSCs either respond to osteogenic stimuli more vividly or, alternatively, CD146(pos) pMSCs present a pMSC subset that is predetermined to differentiate into osteoblasts.

Cell-type specific four-component hydrogel.

In the field of regenerative medicine we aim to develop implant matrices for specific tissue needs. By combining two per se, cell-permissive gel systems with enzymatic crosslinkers (gelatin/transglutaminase and fibrinogen/thrombin) to generate a blend (technical term: quattroGel), an unexpected cell-selectivity evolved. QuattroGels were porous and formed cavities in the cell diameter range, possessed gelation kinetics in the minute range, viscoelastic properties and a mechanical strength appropriate for general cell adhesion, and restricted diffusion. Cell proliferation of endothelial cells, chondrocytes and fibroblasts was essentially unaffected. In contrast, on quattroGels neither endothelial cells formed vascular tubes nor did primary neurons extend neurites in significant amounts. Only chondrocytes differentiated properly as judged by collagen isoform expression. The biophysical quattroGel characteristics appeared to leave distinct cell processes such as mitosis unaffected and favored differentiation of sessile cells, but hampered differentiation of migratory cells. This cell-type selectivity is of interest e.g. during articular cartilage or invertebral disc repair, where pathological innervation and angiogenesis represent adverse events in tissue engineering.

Introducing a combinatorial DNA-toolbox platform constituting defined protein-based biohybrid-materials.

The access to defined protein-based material systems is a major challenge in bionanotechnology and regenerative medicine. Exact control over sequence composition and modification is an important requirement for the intentional design of structure and function. Herein structural- and matrix proteins provide a great potential, but their large repetitive sequences pose a major challenge in their assembly. Here we introduce an integrative "one-vector-toolbox-platform" (OVTP) approach which is fast, efficient and reliable. The OVTP allows for the assembly, multimerization, intentional arrangement and direct translation of defined molecular DNA-tecton libraries, in combination with the selective functionalization of the yielded protein-tecton libraries. The diversity of the generated tectons ranges from elastine-, resilin, silk- to epitope sequence elements. OVTP comprises the expandability of modular biohybrid-materials via the assembly of defined multi-block domain genes and genetically encoded unnatural amino acids (UAA) for site-selective chemical modification. Thus, allowing for the modular combination of the protein-tecton library components and their functional expansion with chemical libraries via UAA functional groups with bioorthogonal reactivity. OVTP enables access to multitudes of defined protein-based biohybrid-materials for self-assembled superstructures such as nanoreactors and nanobiomaterials, e.g. for approaches in biotechnology and individualized regenerative medicine.

Hypoxic conditions during expansion culture prime human mesenchymal stromal precursor cells for chondrogenic differentiation in three-dimensional cultures.

Cell-based approaches using mesenchymal stromal precursor cells (MSCs) for the regeneration of intervertebral discs are attracting increased interest, even though the intervertebral disc is a very demanding environment. Implanted cells eventually face acidic pH, hypoxia, and a lack of nutrients. While the regenerative potential of MSCs for skeletal tissues has been well described, it is still questionable whether human MSCs can be prepared for prolonged survival and proper functioning and whether they can differentiate under the adverse conditions encountered in the disc. Here we examined the influence of hypoxia during expansion and differentiation on the chondrogenesis of MSCs. Chondrogenic differentiation was performed in in situ solidifying gelatin hydrogels, which represent a suitable matrix for delivering and anchoring cells within the disc tissue. To consider limitations in nutrition in the intervertebral disc, differentiation was performed at low cell concentrations in the gelatin hydrogels. Standard high-density micromass cultures served as reference controls. To determine the quality of chondrogenesis we analyzed typical marker molecules such as collagen types I, II, X, Sox-9, MIA, and aggrecan mRNA using RT-qPCR and determined protein deposition by histological stainings and biochemical methods. We could demonstrate that in gelatin-based hydrogels chondrogenic differentiation of human MSCs is possible at low cell concentrations. The quality of chondrogenic differentiation could be improved by hypoxia. Best results were obtained when the entire in vitro process, including MSC expansion and subsequent differentiation, was done under hypoxic conditions. MSCs that were expanded under reduced oxygen tension were primed for a chondrogenic differentiation.

Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes.

This study is intended to optimise expansion and differentiation of cultured human chondrocytes by growth factor application and to identify molecular markers to monitor their differentiation state. We dissected the molecular consequences of matrix release, monolayer, and 3D-alginate culture, growth factor optimised expansion, and re-differentiation protocols by gene expression analysis. Among 19 common cartilage molecules assessed by cDNA array, six proved best to monitor differentiation. Instant down-regulation at release of cells from the matrix was strongest for COL 2A1, fibromodulin, and PRELP while LUM, CHI3L1, and CHI3L2 were expansion-related. Both gene sets reflected the physiologic effects of the most potent growth-inducing (PDGF-BB) and proteoglycan-inducing (BMP-4) factors. Only CRTAC1 expression correlated with 2D/3D switches while the molecular phenotype of native chondrocytes was not restored. The markers and optimised protocols we suggest can help to improve cell therapy of cartilage defects and chondrocyte differentiation from stem cell sources.

Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells.

OBJECTIVE: To compare the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (BMSC) and adipose tissue-derived stromal cells (ATSC), because the availability of an unlimited cell source replacing human chondrocytes could be strongly beneficial for cell therapy, tissue engineering, in vitro drug screening, and development of new therapeutic options to enhance the regenerative capacity of human cartilage. METHODS: Quantitative gene expression of common cartilage and cell interaction molecules was analyzed using complementary DNA array technology and reverse transcription-polymerase chain reaction during optimization of cell differentiation, in order to achieve a molecular phenotype similar to that of chondrocytes in cartilage. RESULTS: The multilineage potential of BMSC and ATSC was similar according to cell morphology and histology, but minor differences in marker gene expression occurred in diverse differentiation pathways. Although chondrogenic differentiation of BMSC and ATSC was indistinguishable in monolayer and remained partial, only BMSC responded (with improved chondrogenesis) to a shift to high-density 3-dimensional cell culture, and reached a gene expression profile highly homologous to that of osteoarthritic (OA) cartilage. CONCLUSION: Hypertrophy of chondrocytes and high matrix-remodeling activity in differentiated BMSC spheroids and in OA cartilage may be the basis for the strong similarities in gene expression profiles between these samples. Differentiated stem cell spheroids represent an attractive tool for use in drug development and identification of drug targets in OA cartilage-like tissue outside the human body. However, optimization of differentiation protocols to achieve the phenotype of healthy chondrocytes is desired for cell therapy and tissue engineering approaches.