Amino Acid-Based Polyphosphorodiamidates with Hydrolytically Labile Bonds for Degradation-Tuned Photopolymers.

Photochemical additive manufacturing technologies can produce complex geometries in short production times and thus have considerable potential as a tool to fabricate medical devices such as individualized patient-specific implants, prosthetics and tissue engineering scaffolds. However, most photopolymer resins degrade only slowly under the mild conditions required for many biomedical applications. Herein we report a novel platform consisting of amino acid-based polyphosphorodiamidate (APdA) monomers with hydrolytically cleavable bonds. The substituent on the α-amino acid can be used as a handle for facile control of hydrolysis rates of the monomers into their endogenous components, namely phosphate and the corresponding amino acid. Furthermore, monomer hydrolysis is considerably accelerated at lower pH values. The monomers underwent thiol-yne photopolymerization and could be 3D structured via multiphoton lithography. Copolymerization with commonly used hydrophobic thiols demonstrates not only their ability to regulate the ambient degradation rate of thiol-yne polyester photopolymer resins, but also desirable surface erosion behavior. Such degradation profiles, in the appropriate time frames, in suitably mild conditions, combined with their low cytotoxicity and 3D printability, render these novel photomonomers of significant interest for a wide range of biomaterial applications.

A Biodegradable Polymeric Matrix for the Repair of Annulus Fibrosus Defects in Intervertebral Discs.

BACKGROUND: Tissue defects in the annulus fibrosus (AF) due to intervertebral disc (IVD) degeneration or after nucleodiscectomy have little self-healing capacity. To prevent progressive degeneration of the IVD, the AF must be repaired. Biological closure has not yet been achieved and is a challenge for the research community. In this study, a scaffold made of absorbable poly (glycolic acid) (PGA) and hyaluronan (HA) that exhibit excellent biocompatibility and cell colonization properties was used to repair AF defects in an ovine model. METHODS: A partial resection was performed in AF in L3/4 or L4/5 of 10 sheep and PGA-HA scaffolds were implanted on the defects (n = 5), while defects in the control group were left untreated (n = 5). Three months post-operation, the lumbar discs were sectioned and stained with hematoxylin and eosin and safranin-O/fast-green. Histological features including proteoglycan content, annular structure, cellular morphology, blood vessel ingrowth and tear/cleft formation were scored using a modified scoring scheme by 3 investigators and evaluated by a pathologist independently. RESULTS: The treated AF exhibited significantly enhanced repair tissue structure with signs of proteoglycan formation compared to the untreated group. The median scores were 4.3 for the treated and 9.8 for the untreated group. Cystic degeneration, perivascular infiltration, inflammation and necrosis were only present in the untreated group. Blood vessel ingrowth and tear/cleft formation were increased, though not significant, in the untreated group while cell morphology was comparable in both groups. CONCLUSION: PGA-HA scaffolds used for AF closure support repair tissue formation in an ovine lumbar disc defect model.

A Novel Method Facilitating the Simple and Low-Cost Preparation of Human Osteochondral Slice Explants for Large-Scale Native Tissue Analysis.

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-ß3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-ß3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.

Comparison of the Translational Potential of Human Mesenchymal Progenitor Cells from Different Bone Entities for Autologous 3D Bioprinted Bone Grafts.

Reconstruction of segmental bone defects by autologous bone grafting is still the standard of care but presents challenges including anatomical availability and potential donor site morbidity. The process of 3D bioprinting, the application of 3D printing for direct fabrication of living tissue, opens new possibilities for highly personalized tissue implants, making it an appealing alternative to autologous bone grafts. One of the most crucial hurdles for the clinical application of 3D bioprinting is the choice of a suitable cell source, which should be minimally invasive, with high osteogenic potential, with fast, easy expansion. In this study, mesenchymal progenitor cells were isolated from clinically relevant human bone biopsy sites (explant cultures from alveolar bone, iliac crest and fibula; bone marrow aspirates; and periosteal bone shaving from the mastoid) and 3D bioprinted using projection-based stereolithography. Printed constructs were cultivated for 28 days and analyzed regarding their osteogenic potential by assessing viability, mineralization, and gene expression. While viability levels of all cell sources were comparable over the course of the cultivation, cells obtained by periosteal bone shaving showed higher mineralization of the print matrix, with gene expression data suggesting advanced osteogenic differentiation. These results indicate that periosteum-derived cells represent a highly promising cell source for translational bioprinting of bone tissue given their superior osteogenic potential as well as their minimally invasive obtainability.

Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades.

Intervertebral disc (IVD) herniation and degeneration is a major source of back pain. In order to regenerate a herniated and degenerated disc, closure of the anulus fibrosus (AF) is of crucial importance. For molecular characterization of AF, genome-wide Affymetrix HG-U133plus2.0 microarrays of native AF and cultured cells were investigated. To evaluate if cells derived from degenerated AF are able to initiate gene expression of a regenerative pattern of extracellular matrix (ECM) molecules, cultivated cells were stimulated with bone morphogenetic protein 2 (BMP2), transforming growth factor ß1 (TGFß1) or tumor necrosis factor-α (TNFα) for 24 h. Comparative microarray analysis of native AF tissues showed 788 genes with a significantly different gene expression with 213 genes more highly expressed in mild and 575 genes in severe degenerated AF tissue. Mild degenerated native AF tissues showed a higher gene expression of common cartilage ECM genes, whereas severe degenerated AF tissues expressed genes known from degenerative processes, including matrix metalloproteinases (MMP) and bone associated genes. During monolayer cultivation, only 164 differentially expressed genes were found. The cells dedifferentiated and altered their gene expression profile. RTD-PCR analyses of BMP2- and TGFß1-stimulated cells from mild and severe degenerated AF tissue after 24 h showed an increased expression of cartilage associated genes. TNFα stimulation increased MMP1, 3, and 13 expression. Cells derived from mild and severe degenerated tissues could be stimulated to a comparable extent. These results give hope that regeneration of mildly but also strongly degenerated disc tissue is possible.

Proliferation, Migration, and ECM Formation Potential of Human Annulus Fibrosus Cells Is Independent of Degeneration Status.

OBJECTIVE: The objective was to evaluate the proliferating, migratory and extracellular matrix (ECM) forming potential of annulus fibrosus cells derived from early (edAFC) or advanced (adAFC) degenerative tissue and their usability as a possible cell source for regenerative approaches for AF closure. DESIGN: EdAFC (n = 5 Pfirrman score of 2-3) and adAFC (n = 5 Pfirrman score of 4-5) were isolated from tissue of patients undergoing spine stabilizing surgery. Cell migration on stimulation with human serum (HS), platelet-rich plasma (PRP), and transforming growth factor ß-3 (TGFB3) was assessed by migration assay and proliferation was assessed on stimulation with HS. Induction of ECM synthesis was evaluated by gene expression analysis of AF-related genes in three-dimensional scaffold cultures that have been stimulated with 5% PRP or 10 ng/mL TGFB3 and histologically by collagen type I, type II, alcian blue, and safranin-O staining. RESULTS: EdAFC and adAFC were significantly attracted by 10% HS and 5% PRP. Additionally, both cell groups proliferated under stimulation with HS. Stimulation with 10 ng/mL TGFB3 showed significant induction of gene expression of collagen type II and aggrecan, while 5% PRP decreased the expression of collagen type I. Both cell groups showed formation of AF-like ECM after stimulation with TGFB3, whereas stimulation with PRP did not. CONCLUSIONS: Our study demonstrated that AF cells retain their potential for proliferation, migration, and ECM formation independent of the degeneration status of the tissue. Proliferation, migration, and ECM synthesis of the endogenous AF cells can be supported by different supplements. Hence, endogenous AF cells might be a suitable cell source for a regenerative repair approaches.

Cardiac Extracellular Vesicles (EVs) Released in the Presence or Absence of Inflammatory Cues Support Angiogenesis in Different Manners.

Cells release extracellular vesicles (EVs) to communicate in a paracrine manner with other cells, and thereby influence processes, such as angiogenesis. The conditioned medium of human cardiac-derived adherent proliferating (CardAP) cells was recently shown to enhance angiogenesis. To elucidate whether their released EVs are involved, we isolated them by differential centrifugation from the conditioned medium derived either in the presence or absence of a pro-inflammatory cytokine cocktail. Murine recipient cells internalized CardAP-EVs as determined by an intracellular detection of human proteins, such as CD63, by a novel flow cytometry method for studying EV-cell interaction. Moreover, endothelial cells treated for 24 h with either unstimulated or cytokine stimulated CardAP-EVs exhibited a higher tube formation capability on Matrigel. Interestingly, unstimulated CardAP-EVs caused endothelial cells to release significantly more vascular endothelial growth factor and interleukin (IL)-6, while cytokine stimulated CardAP-EVs significantly enhanced the release of IL-6 and IL-8. By nCounter® miRNA expression assay (NanoString Technologies) we identified microRNA 302d-3p to be enhanced in unstimulated CardAP-EVs compared to their cytokine stimulated counterparts, which was verified by quantitative polymerase chain reaction. This study demonstrates that both CardAP-EVs are pro-angiogenic by inducing different factors from endothelial cells. This would allow to select potent targets for a safe and efficient therapeutic application.

Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-Like Cells for Allogeneic Cell Therapies.

Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, we evaluated their inductive and modulatory capacity regarding immune responses and underlying key mechanisms in vitro. For this, cryopreserved hAACs were either cultured in the presence of interferon-gamma (IFNγ) or left unstimulated. The expression of characteristic mesenchymal stromal cell markers (CD29, CD44, CD73, CD105, CD166) was revealed by flow cytometry that also highlighted a predominant negativity for CD90. A low immunogeneic phenotype in an inflammatory milieu was shown by lacking expression of co-stimulatory molecules and upregulation of the inhibitory ligands PD-L1 and PD-L2, despite de novo expression of HLA-DR. Co-cultures of hAACs with allogeneic peripheral blood mononuclear cells, proved their low immunogeneic state by absence of induced T cell proliferation and activation. Additionally, elevated levels of IL-1ß, IL-33, and IL-10 were detectable in those cell culture supernatants. Furthermore, the immunomodulatory potential of hAACs was assessed in co-cultures with αCD3/αCD28-activated peripheral blood mononuclear cells. Here, a strong inhibition of T cell proliferation and reduction of pro-inflammatory cytokines (IFNγ, TNFα, TNFß, IL-17A, IL-2) were observable after pre-stimulation of hAACs with IFNγ. Transwell experiments confirmed that mostly soluble factors are responsible for these suppressive effects. We were able to identify indolamin-2,3-dioxygenase (IDO) as a potential key player through a genome-wide gene expression analysis and could demonstrate its involvement in the observed immunological responses. While the application of blocking antibodies against both PD-1 ligands did not affect the immunomodulation by hAACs, 1-methyl-L-tryptophan as specific inhibitor of IDO was able to restore proliferation and to lower apoptosis of T cells. In conclusion, hAACs represent a cardiac-derived mesenchymal stromal-like cell type with a high potential for the application in an allogeneic setting, since they do not trigger T cell responses and even increase their immunomodulatory potential in inflammatory environments.

Effect of inoculum density on human-induced pluripotent stem cell expansion in 3D bioreactors.

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.

CCL25-Supplemented Hyaluronic Acid Attenuates Cartilage Degeneration in a Guinea Pig Model of Knee Osteoarthritis.

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. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1723-1729, 2019.

Online measurement of oxygen enables continuous noninvasive evaluation of human-induced pluripotent stem cell (hiPSC) culture in a perfused 3D hollow-fiber bioreactor.

For clinical and/or pharmaceutical use of human-induced pluripotent stem cells (hiPSCs), large cell quantities of high quality are demanded. Therefore, we combined the expansion of hiPSCs in closed, perfusion-based 3D bioreactors with noninvasive online monitoring of oxygen as culture control mechanism. Bioreactors with a cell compartment volume of 3 or 17 ml were inoculated with either 10 × 106 or 50 × 106 cells, and cells were expanded over 15 days with online oxygen and offline glucose and lactate measurements being performed. The CellTiter-Blue® Assay was performed at the end of the bioreactor experiments for indirect cell quantification. Model simulations enabled an estimation of cell numbers based on kinetic equations and experimental data during the 15-day bioreactor cultures. Calculated oxygen uptake rates (OUR), glucose consumption rates (GCR), and lactate production rates (LPR) revealed a highly significant correlation (p < 0.0001). Oxygen consumption, which was measured at the beginning and the end of the experiment, showed a strong culture growth in line with the OUR and GCR data. Furthermore, the yield coefficient of lactate from glucose and the OUR to GCR ratio revealed a shift from nonoxidative to oxidative metabolism. The presented results indicate that oxygen is equally as applicable as parameter for hiPSC expansion as glucose while providing an accurate real-time impression of hiPSC culture development. Additionally, oxygen measurements inform about the metabolic state of the cells. Thus, the use of oxygen online monitoring for culture control facilitates the translation of hiPSC use to the clinical setting.

Biomimetic sulfated polyethylene glycol hydrogel inhibits proteoglycan loss and tumor necrosis factor-α-induced expression pattern in an osteoarthritis in vitro model.

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.

Dose-Dependent Effect of Mesenchymal Stromal Cell Recruiting Chemokine CCL25 on Porcine Tissue-Engineered Healthy and Osteoarthritic Cartilage.

Thymus-expressed chemokine (CCL25) is a potent cell attractant for mesenchymal stromal cells, and therefore it is a candidate for in situ cartilage repair approaches focusing on the recruitment of endogenous repair cells. However, the influence of CCL25 on cartilage is unknown. Accordingly, in this study, we investigated the effect of CCL25 on tissue-engineered healthy and osteoarthritic cartilage. Porcine chondrocytes were cultured in a three-dimensional (3D) micromass model that has been proven to mimic key-aspects of human cartilage and osteoarthritic alterations upon stimulation with tumor necrosis factor-α (TNF-α). Micromass cultures were stimulated with CCL25 (0, 0.05, 0.5, 5, 50, 500 nmol/L) alone or in combination with 0.6 nmol/L TNF-α for seven days. Effects were evaluated by life/dead staining, safranin O staining, histomorphometrical analysis of glycosaminoglycans (GAGs), collagen type II (COL2A1) real-time RT-PCR and Porcine Genome Array analysis. 500 nmol/L CCL25 led to a significant reduction of GAGs and COL2A1 expression and induced the expression of matrix metallopeptidases (MMP) 1, MMP3, early growth response protein 1 (EGR1), and superoxide dismutase 2 (SOD2). In concentrations lower than 500 nmol/L, CCL25 seems to be a candidate for in situ cartilage repair therapy approaches.

CCL25 chemokine-guided stem cell attraction: an assessment of possible benefits and risks.

Due to its chemoattraction potential on mesenchymal stromal cells of the CCL25/CCR9 axis, local application of CCL25 to severely damaged tissues may be a promising approach for regenerative therapies. Analysis of the given data revealed that CCL25/CCR9 signaling has a crucial role in regulation of an adult immune homeostasis. CCR9 expression variations resulted in dysfunctional immune response in colitis, rheumatoid arthritis and endometriosis. Regarding oncology, different neoplastic tissues exploit CCL25-dependent CCR9 signaling for either local proliferation or migration processes. The CCR9 pathway likely can trigger crosstalk between the Akt and NOTCH pathway and thus participate in the regulation of the neoplastic behavior. In conclusion, the designated application-tissue requires precise molecular analysis of possible CCR9 expression due to its proto-oncogenic characteristics.

Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells.

BACKGROUND: The lifetime prevalence of degenerative disc disease is dramatically high. Numerous investigations on disc degeneration have been performed on cells from annulus fibrosus (AF) and nucleus pulposus (NP) of the intervertebral disc (IVD) in cell culture experiments utilising a broad variety of basal culture media. Although the basal media differ in nutrient formulation, it is not known whether the choice of the basal media itself has an impact on the cell's behaviour in vitro. In this study, we evaluated the most common media used for monolayer expansion of AF and NP cells to set standards for disc cell culture. METHODS: Human AF and NP cells were isolated from cervical discs. Cells were expanded in monolayer until passage P2 using six different common culture media containing alpha-Minimal Essential Medium (alpha-MEM), Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F-12 medium (Ham's F-12) as single medium or in a mixture of two media (alpha/F-12, DMEM/alpha, DMEM/F-12). Cell morphology, cell growth, glycosaminoglycan production and quantitative gene expression of cartilage- and IVD-related markers aggrecan, collagen type II, forkhead box F1 and keratin 18 were analysed. Statistical analysis was performed with two-way ANOVA testing and Bonferroni compensation. RESULTS: AF and NP cells were expandable in all tested media. Both cell types showed similar cell morphology and characteristics of dedifferentiation known for cultured disc cells independently from the media. However, proceeding culture in Ham's F-12 impeded cell growth of both AF and NP cells. Furthermore, the keratin 18 gene expression profile of NP cells was changed in alpha-MEM and Ham's F-12. CONCLUSION: The impact of the different media itself on disc cell's behaviour in vitro was low. However, AF and NP cells were only robust, when DMEM was used as single medium or in a mixture (DMEM/alpha, DMEM/F-12). Therefore, we recommend using these media as standard medium for disc cell culture. Our findings are valuable for the harmonisation of preclinical study results and thereby push the development of cell therapies for clinical treatment of disc degeneration.

Quality Assessment of Surgical Disc Samples Discriminates Human Annulus Fibrosus and Nucleus Pulposus on Tissue and Molecular Level.

A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration.

Hyaluronic Acid Influence on Normal and Osteoarthritic Tissue-Engineered Cartilage.

The aim of this study is to identify gene expression profiles associated with hyaluronic acid (HA) treatment of normal and osteoarthritis (OA)-like tissue-engineered cartilage. 3D cartilage micromasses were treated with tumour necrosis factor-α (TNF-α) (OA-inducer) and/or HA for 7 days. Viability was examined by PI/FDA staining. To document extracellular matrix (ECM) formation, glycosaminoglycans (GAG) were stained with Safranin-O and cartilage-specific type II collagen was detected immunohistochemically. Genome-wide gene expression was determined using microarray analysis. Normal and OA-like micromasses remained vital and showed a spherical morphology and homogenous cell distribution regardless of the treatment. There was no distinct difference in immunolabeling for type II collagen. Safranin-O staining demonstrated a typical depletion of GAG in TNF-α-treated micromasses (-73%), although the extent was limited in the presence of HA (-39%). The microarray data showed that HA can influence the cartilage metabolism via upregulation of TIMP3 in OA-like condition. The upregulation of VEGFA and ANKRD37 genes implies a supportive role of HA in cartilage maturation and survival. The results of this study validate the feasibility of the in vitro OA model for the investigation of HA. On the cellular level, no inhibiting or activating effect of HA was shown. Microarray data demonstrated a minor impact of HA on gene expression level.

Delayed release of chemokine CCL25 with bioresorbable microparticles for mobilization of human mesenchymal stem cells.

Chemokines are guiding cues for directional trafficking of mesenchymal stem cells (MSC) upon injury and local chemokine delivery at injury sites is an up-to-date strategy to potentiate and prolong recruitment of MSC. In this study we present the chemokine CCL25, also referred to as thymus-expressed chemokine, to mobilize human MSC along positive but not along negative gradients. We hence proceeded to design a biodegradable and injectable release device for CCL25 on the basis of poly(lactic-co-glycolic acid) (PLGA). The conducted studies had the objective to optimize PLGA microparticle fabrication by varying selected formulation parameters, such as polymer type, microparticle size and interior phase composition. We found that microparticles of DV,50∼75 µm and fabricated using end-capped polymers, BSA as carrier protein and vortex mixing to produce the primary emulsion yielded high chemokine loading and delayed CCL25 release. To determine bioactivity, we investigated CCL25 released during the microparticle erosion phase and showed that deacidification of the release medium was required to induce significant MSC mobilization. The designed PLGA microparticles represent an effective and convenient off-the-shelf delivery tool for the delayed release of CCL25. However, continuative in vivo proof-of-concept studies are required to demonstrate enhanced recruitment of MSC and/or therapeutical effects in response to CCL25 release microparticles. STATEMENT OF SIGNIFICANCE: With the discovery of chemokines, particularly CXCL12, as stimulators of stem cell migration, the development of devices that release CXCL12 has proceeded quickly in the last few years. In this manuscript we introduce CCL25 as chemokine to induce mobilization of human MSC. This study proceeds to demonstrate how selection of key formulation parameters of CCL25 loading into PLGA microparticles exerts considerable influence on CCL25 release. This is important for a broad range of efforts in in situ tissue engineering where the candidate chemokine and the delivery device need to be selected carefully. The use of such a cell-free CCL25 release device may provide a new therapeutic option in regenerative medicine.

The atrial appendage as a suitable source to generate cardiac-derived adherent proliferating cells for regenerative cell-based therapies.

Cardiac-derived adherent proliferating (CardAP) cells obtained from endomyocardial biopsies (EMBs) with known anti-fibrotic and pro-angiogenic properties are good candidates for the autologous therapy of end-stage cardiac diseases such as dilated cardiomyopathy. However, due to the limited number of CardAP cells that can be obtained from EMBs, our aim is to isolate cells with similar properties from other regions of the heart with comparable tissue architecture. Here, we introduce the atrial appendage as a candidate region. Atrial appendage-derived cells were sorted with CD90 microbeads to obtain a CD90low cell population, which were subsequently analysed for their surface marker and gene expression profiles via flow cytometry and micro array analysis. Enzyme-linked immunosorbent assays for vascular endothelial growth factor and interleukin-8 as well as tube formation assays were performed to investigate pro-angiogenic properties. Furthermore, growth kinetic assays were performed to estimate the cell numbers needed for cell-based products. Microarray analysis revealed the expression of numerous pro-angiogenic genes and strong similarities to CardAP cells with which they also share expression levels of defined surface antigens, that is, CD29+ , CD44+ , CD45- , CD73+ , CD90low , CD105+ , and CD166+ . High secretion levels of vascular endothelial growth factor and interleukin-8 as well as improved properties of vascular structures in vitro could be detected. Based on growth parameters, cell dosages for the treatment of more than 250 patients are possible using one appendage. These results lead to the conclusion that isolating cells with regenerative characteristics from atrial appendages is feasible and permits further investigations towards allogenic cell-based therapies.

Characterization of single cell derived cultures of periosteal progenitor cells to ensure the cell quality for clinical application.

For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations. For an efficient and approved application in bone or cartilage regeneration the clarification of this question is of interest. Therefore, 83 different clonal cultures of freshly isolated human periosteal cells derived from mastoid periosteum biopsies of 4 donors were generated and growth rates calculated. Differentiation capacities of 51 clonal cultures towards the osteogenic, the chondrogenic, and the adipogenic lineage were investigated. Histological and immunochemical stainings showed that 100% of the clonal cultures differentiated towards the osteogenic lineage, while 94.1% demonstrated chondrogenesis, and 52.9% could be stimulated to adipogenesis. For osteogenesis real-time polymerase chain reaction (PCR) of BGLAP and RUNX2 and for adipogenesis of FABP4 and PPARG confirmed the results. Overall, 49% of the cells exhibited a tripotent potential, 45.1% showed a bipotent potential (without adipogenic differentiation), 3.9% bipotent (without chondrogenic differentiation), and 2% possessed a unipotent osteogenic potential. In FACS analyses, no differences in the marker profile of undifferentiated clonal cultures with bi- and tripotent differentiation capacity were found. Genome-wide microarray analysis revealed 52 differentially expressed genes for clonal subpopulations with or without chondrogenic differentiation capacity, among them DCN, NEDD9, TGFBR3, and TSLP. For clinical applications of periosteal cells in bone regeneration all cells were inducible. For a chondrogenic application a fraction of 6% of the mixed population could not be induced.