A bead-based multiplex assay covering all coronaviruses pathogenic for humans for sensitive and specific surveillance of SARS-CoV-2 humoral immunity.

Serological assays measuring antibodies against SARS-CoV-2 are key to describe the epidemiology, pathobiology or induction of immunity after infection or vaccination. Of those, multiplex assays targeting multiple antigens are especially helpful as closely related coronaviruses or other antigens can be analysed simultaneously from small sample volumes, hereby shedding light on patterns in the immune response that would otherwise remain undetected. We established a bead-based 17-plex assay detecting antibodies targeting antigens from all coronaviruses pathogenic for humans: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV strains 229E, OC43, HKU1, and NL63. The assay was validated against five commercial serological immunoassays, a commercial surrogate virus neutralisation test, and a virus neutralisation assay, all targeting SARS-CoV-2. It was found to be highly versatile as shown by antibody detection from both serum and dried blot spots and as shown in three case studies. First, we followed seroconversion for all four endemic HCoV strains and SARS-CoV-2 in an outbreak study in day-care centres for children. Second, we were able to link a more severe clinical course to a stronger IgG response with this 17-plex-assay, which was IgG1 and IgG3 dominated. Finally, our assay was able to discriminate recent from previous SARS-CoV-2 infections by calculating the IgG/IgM ratio on the N antigen targeting antibodies. In conclusion, due to the comprehensive method comparison, thorough validation, and the proven versatility, our multiplex assay is a valuable tool for studies on coronavirus serology.

Serum interleukin-6, procalcitonin, and C-reactive protein at hospital admission can identify patients at low risk for severe COVID-19 progression.

Background: COVID-19 can show a variable course, from asymptomatic infections to acute respiratory failure and death. For efficient allocation of resources, patients should be stratified according to their risk for a severe course as early as possible. Methods: 135 hospitalized patients with COVID-19 pneumonia at four German hospitals were prospectively included in this observational study. A standardized clinical laboratory profile was taken at hospital admission and a panel of serum markers with possible roles in the COVID-associated cytokine storm were also determined. 112 patients could be evaluated. The primary endpoint of ventilator requirement or death within 30 days of symptom onset was met by 13 patients. Results: Serum elevations of interleukin-6 (IL-6), procalcitonin (PCT), and C-reactive protein (CRP) at hospital admission were each highly significantly (p < 0.001) associated with ventilator requirement/death within 30 days of symptom onset. With a sensitivity of 92% and a specificity of 65-67%, IL-6 ≥ 52.8 pg/ml, PCT ≥ 0.11 ng/ml, and CRP ≥ 71.1 mg/L were predictive of a severe course of COVID-19. Positive likelihood ratios were between 2.6-2.8 and negative likelihood ratios were between 0.11-0.13 for these three markers. Conclusion: Negative likelihood ratios indicate that IL-6, PCT, and CRP at hospital admission can be used for identifying patients at low risk for severe COVID-19 progression.

Association between different dimensions of anger and symptoms of post-traumatic stress disorder in at-risk cardiovascular patients during the COVID-19 pandemic.

Introduction: The common connecting factor between PTSD and cardiovascular diseases lies in the disruption of the stress processing system. The COVID-19 pandemic has led to an increase in stress levels worldwide. Due to the life-threatening situation of affected risk patients, this also led to the accumulation of post-traumatic stress symptoms (PTSS). The influence of anger on cardiovascular diseases has hardly been investigated so far. The focus of this study is on anger regulation in cardiovascular risk patients. The COVID-19 pandemic is considered as an additional stressor in this study, but not as a separate entity. The hypothesis is that individuals with inward anger are more prone to post-traumatic stress disorder (PTSD). Methods: As part of the routine examination, all patients who were hospitalized between January 1st, 2021 and May 31st, 2022 with high-risk cardiovascular diseases were included. A total of N = 153 (84.1%) subjects participated in the study. On admission, anger (STAXI-2) and PTSD (PCL-5) were assessed using questionnaires. The relationship between different domains of anger and PTSS was examined. Results: Inwardly directed anger was more pronounced in this population than in a standard sample (+1 SD) and had a significant impact on the presence of PTSD (B = -0.72, p < 0.001). Additionally, correlations were found between inward-directed anger and PTSD, as well as all other anger expressions studied and the PTSD total score. Discussion: It can be assumed that anger and its regulation are relevant factors for both cardiac diseases and PTSD. The study results can be used for prevention, rehabilitation and therapeutic measures. However, the impact of inner anger on PTSD is theoretical and based on statistical testing. A confirmatory longitudinal study is needed to substantiate these results.

Blends of gelatin and hyaluronic acid stratified by stereolithographic bioprinting approximate cartilaginous matrix gradients.

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.

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.

Photopolymerizable gelatin and hyaluronic acid for stereolithographic 3D bioprinting of tissue-engineered cartilage.

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.

Characterization of plasma fibronectin for migration, proliferation, and differentiation on human articular chondrocytes.

Plasma fibronectin (pFN) plays a crucial role in wound healing by binding to integrins and inducing cell migration. It is known to induce the migration and proliferation of mesenchymal progenitor cells in vitro, which play a key role during microfracture in cartilage repair. Endogenous chondrocytes from the native cartilage of the defect rim might aid in cartilage repair. In this study, the effect of pFN on proliferation, migration, and differentiation was tested on human articular chondrocytes. Results showed that treatment with pFN increased the migration of chondrocytes in a range of 1-30 µg/ml as tested with no effect on proliferation. TGFß3-induced chondrogenesis was not affected by pFN. Especially, gene expression of matrix metalloproteinases was not increased by pFN. Plasma FN fragmentation due to storage conditions could be excluded by SDS-PAGE. Moreover, bioactivity of pFN did not alter during storage at 4°C and 40°C for up to 14 days. Taken together, pFN induces the migration but not proliferation of human articular chondrocytes with no inhibitory effect on chondrogenic differentiation. Additionally, no loss of activity or fragmentation of pFN was observed after lyophilization and storage, making pFN an interesting bioactive factor for chondrocyte recruitment.

Chemokine CCL25 Induces Migration and Extracellular Matrix Production of Anulus Fibrosus-Derived Cells.

Intervertebral disc degeneration is a major source of back pain. For intervertebral disc regeneration after herniation a fast closure of anulus fibrosus (AF) defects is crucial. Here, the use of the C-C motif chemokine ligand 25 (CCL)25 in comparison to differentiation factors such as transforming growth factor (TGF)ß3, bone morphogenetic protein (BMP)2, BMP7, BMP12, and BMP14 (all in concentrations of 10, 50 and 100 ng/mL) was tested in an in vitro micro mass pellet model with isolated and cultivated human AF-cells (n = 3) to induce and enhance AF-matrix formation. The pellets were differentiated (serum-free) with supplementation of the factors. After 28 days all used factors induced proteoglycan production (safranin O staining) and collagen type I production (immunohistochemical staining) in at least one of the tested concentrations. Histomorphometric scoring revealed that TGFß3 delivered the strongest induction of proteoglycan production in all three concentrations. Furthermore, it was the only factor able to facilitate collagen type II production, even higher than in native tissue samples. CCL25 was also able to induce proteoglycan and collagen type I production comparable to several BMPs. CCL25 could additionally induce migration of AF-cells in a chemotaxis assay and therefore possibly aid in regeneration processes after disc herniation by recruiting AF-cells.

Proliferation, migration and differentiation potential of human mesenchymal progenitor cells derived from osteoarthritic subchondral cancellous bone.

Background: For regenerative therapies in the orthopedic field, one prerequisite for therapeutic success in the treatment of cartilage defects is the potential of body's own cells to migrate, proliferate and differentiate into functional cells. While this has been demonstrated for mesenchymal stem and progenitor cells (MPC) from healthy tissue sources, the potential of cells from degenerative conditions is unclear. In this study the regenerative potential of MPC derived from subchondral cancellous bone with diagnosed osteoarthritis is evaluated in vitro. Methods: OaMPC isolated from bone chips of three individual patients with Kellgren grade 3 osteoarthritis were characterized by analysis of cell surface antigen pattern. Cell proliferation was evaluated by doubling time and population doubling rate. Cell migration was assessed using a multi-well migration assay. Multi-lineage potential was evaluated by histological staining of adipogenic, osteogenic and chondrogenic markers. In addition, chondrogenic differentiation was verified by qPCR. Results: OaMPC showed a stable proliferation and a typical surface antigen pattern known from mesenchymal stem cells. Cell migration of oaMPC can be induced by human blood serum. OaMPC were capable of adipogenic, osteogenic and chondrogenic differentiation comparable to MPC derived from healthy conditions. Conclusion: OaMPC derived from knee joints affected by osteoarthritic conditions showed regeneration potential regarding migration, proliferation and chondrogenic differentiation. This suggests that oaMPC are able to contribute to cartilage repair tissue formation.

Effect of serum and platelet-rich plasma on human early or advanced degenerative meniscus cells.

PURPOSE: The purpose of this in vitro study was to evaluate the migratory, proliferating, and extracellular matrix (ECM) forming effect of human serum (HS) and platelet-rich plasma (PRP) on meniscus cells derived from human knees with early or advanced degenerative changes. MATERIALS AND METHODS: Medial menisci from knees with early degenerative changes (n = 5; mean Kellgren score of 1) undergoing arthroscopic meniscal surgery and advanced degenerative changes (n = 5; mean Kellgren score of 4) undergoing total knee replacement were collected. Cell migration and proliferation upon stimulation with HS and PRP were assessed by migration and proliferation assays. Induction of meniscal ECM was evaluated histologically by hematoxylin and eosin, collagen type I, and alcian blue staining and by gene expression analysis of meniscus-related genes in pellets that have been stimulated with 10% HS or 5% PRP. RESULTS: Meniscal cells from knees with early and advanced degenerative changes were significantly attracted by 2.5%-30% PRP or 10% HS. Cell proliferation was significantly increased upon stimulation with 10% HS or 5% PRP. Both cell groups showed the formation of a well-structured, meniscus-like ECM after stimulation with 10% HS, whereas stimulation with 5% PRP led to inhomogeneous, more fibrous ECM. Stimulation with 10% HS showed a significant induction of aggrecan and COMP, while 5% PRP showed no inducing effect. CONCLUSIONS: Only stimulation with HS showed the formation of meniscal ECM as well as cell proliferating and migratory effects on meniscal cells derived from knees with early or advanced degenerative changes. Thus, we suggest that the selected stimulating factor itself and not the status of the knee may primarily affect repair processes. HS may have a potential to augment in meniscal repair procedures.

Effect of Human Serum and 2 Different Types of Platelet Concentrates on Human Meniscus Cell Migration, Proliferation, and Matrix Formation.

PURPOSE: To evaluate the effect of 10% human serum (HS), 5% platelet-rich plasma (PRP), and 5% autologous conditioned plasma (ACP) on migration, proliferation, and extracellular matrix (ECM) synthesis of human meniscus cells. METHODS: Cell migration and proliferation on stimulation with HS, PRP, and ACP were assessed by chemotaxis assays and measurement of genomic DNA content. Meniscus cells were cultivated in pellets stimulated with 10% HS, 5% PRP, or 5% ACP. Meniscal ECM formation was evaluated by histochemical staining of collagen type I, type II, and proteoglycans and by analysis of fibrochondrocyte marker gene expression. RESULTS: Human meniscus cells were significantly attracted by all 3 blood-derived products (10% HS and 5% ACP: P = .0001, 5% PRP: P = .0002). Cell proliferation at day 9 was significantly increased on stimulation with 10% HS (P = .0001) and 5% PRP (P = .0002) compared with 5% ACP and controls. Meniscus cell pellet cultures showed the formation of a well-structured meniscal ECM with deposition of collagen type I, type II, and proteoglycans on stimulation with 10% HS, whereas 5% PRP or 5% ACP resulted in the formation of an inhomogeneous and more fibrous ECM. Stimulation with 10% HS and 5% ACP showed a significant induction of fibrochondrocyte marker genes such as aggrecan (HS: P = .0002, ACP: P = .0147), cartilage oligomeric matrix protein (HS: P = .0002, ACP: P = .0005), and biglycan (HS: P = .0002, ACP: P = .0003), whereas PRP showed no inducing effect. CONCLUSIONS: Among all tested blood-derived products, only stimulation with HS showed the formation of a meniscal ECM as well as positive cell proliferating and migrating effects in vitro. Regarding a potential biological repair of nonvascular meniscus lesions, our results may point toward the use of HS as a beneficial augment in regenerative meniscus repair approaches. CLINICAL RELEVANCE: Our findings may suggest that HS might be a beneficial augment for meniscus repair.

Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

PURPOSE: To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. METHODS: Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. RESULTS: MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P < .0001; and PRP-C, P < .0001) stimulated migration of MPCs. All platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. CONCLUSIONS: Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP preparations did induce such differentiation. These findings suggest differing outcomes with PRP treatment in stem cell-based cartilage repair. CLINICAL RELEVANCE: Our findings may help to explain the variability of results in studies examining the use of PRP clinically.

Fibronectin stimulates migration and proliferation, but not chondrogenic differentiation of human subchondral progenitor cells.

AIMS: To evaluate the impact of human plasma-derived fibronectin (FN) on human subchondral mesenchymal progenitor cells regarding cell migration, proliferation, and chondrogenic differentiation. MATERIALS & METHODS: Human subchondral mesenchymal progenitor cells were analyzed for their migration capacity upon treatment with human plasma-derived FN. Proliferation activity was evaluated by DNA content. For chondrogenesis, cells were cultured in high-density pellet cultures in the presence of FN, TGFß3, and a combination thereof. RESULTS: Treatment of progenitors with FN significantly increased the number of migrating cells and elevated proliferative activity. Histological staining indicated formation of an extracellular matrix with type II collagen. Gene expression analysis gave no evidence for chondrogenic differentiation mediated by FN, but revealed a significant induction of type II collagen expression. CONCLUSION: FN has a potential to recruit human subchondral mesenchymal progenitor cells, possibly supporting proliferation and matrix assembly in cartilage repair procedures using bioactive implants after microfracture treatment.

A P19 and P19CL6 cell-based complementary approach to determine paracrine effects in cardiac tissue engineering.

The negligible self-repair potential of the myocardium has led to cell-based tissue engineering approaches to restore heart function. There is more and more consensus that, in addition to cell development, paracrine effects in particular play a pivotal role in the repair of heart tissue. Here, we present two complementary murine P19 and P19CL6 embryonic carcinoma cell-based in vitro test approaches to study the potential of repair cells and the factors secreted by these cells to induce cardiomyogenesis. P19 cells were 3-dimensionally cultured in hanging drops and P19CL6 cells in a monolayer. Both systems, capable of inducible differentiation towards the cardiomyogenic lineage shown by the appearance of beating cells, the expression of connexin 43 and cardiac troponins T and I, were used to test the cardiomyogenesis-inducing potential of human cardiac-derived adherent proliferating (CardAP) cells, which are candidates for heart repair. CardAP cells in coculture as well as CardAP cell-conditioned medium initiated beating in P19 cells, depending on the cell composition and concentration of the medium. CardAP cell-dependent beating was not observed in P19CL6 cultures, but connexin 43 and cardiac troponin formation as well as expression of GATA-binding protein 4 indicated the dose-dependent stimulatory cardiomyogenic effect of human CardAP cells. In summary, in different ways, P19 and P19CL6 cells have shown their capability to detect paracrine effects of human CardAP cells. In a complementary approach, they could be beneficial for determining the stimulatory cardiomyogenic potential of candidate cardiac-repair cells in vitro.

Initial boost release of transforming growth factor-ß3 and chondrogenesis by freeze-dried bioactive polymer scaffolds.

In cartilage regeneration, bio-activated implants are used in stem and progenitor cell-based microfracture cartilage repair procedures. Our aim was to analyze the chondrogenic potential of freeze-dried resorbable polymer-based polyglycolic acid (PGA) scaffolds bio-activated with transforming growth factor-ß3 (TGFB3) on human subchondral mesenchymal progenitor cells known from microfracture. Progenitor cells derived from femur heads were cultured in the presence of freeze-dried TGFB3 in high-density pellet culture and in freeze-dried TGFB3-PGA scaffolds for chondrogenic differentiation. Progenitor cell cultures in PGA scaffolds as well as pellet cultures with and without continuous application of TGFB3 served as controls. Release studies showed that freeze-dried TGFB3-PGA scaffolds facilitate a rapid, initial boost-like release of 71.5% of TGFB3 in the first 10 h. Gene expression analysis and histology showed induction of typical chondrogenic markers like type II collagen and formation of cartilaginous tissue in TGFB3-PGA scaffolds seeded with subchondral progenitor cells and in pellet cultures stimulated with freeze-dried TGFB3. Chondrogenic differentiation in freeze-dried TGFB3-PGA scaffolds was comparable to cultures receiving TGFB3 continuously, while non-stimulated controls did not show chondrogenesis during prolonged culture for 14 days. These results suggest that bio-activated, freeze-dried TGFB3-PGA scaffolds have chondrogenic potential and are a promising tool for stem cell-mediated cartilage regeneration.

Effects of initial boost with TGF-beta 1 and grade of intervertebral disc degeneration on 3D culture of human annulus fibrosus cells.

BACKGROUND: Three-dimensional (3D) culture in porous biomaterials as well as stimulation with growth factors are known to be supportive for intervertebral disc cell differentiation and tissue formation. Unless sophisticated releasing systems are used, however, effective concentrations of growth factors are maintained only for a very limited amount of time in in vivo applications. Therefore, we investigated, if an initial boost with transforming growth factor-beta 1 (TGF-beta 1) is capable to induce a lasting effect of superior cartilaginous differentiation in slightly and severely degenerated human annulus fibrosus (AF) cells. METHODS: Human AF tissue was harvested during surgical treatment of six adult patients with lumbar spinal diseases. Grading of disc degeneration was performed with magnet resonance imaging. AF cells were isolated and expanded in monolayer culture and rearranged three-dimensionally in a porous biomaterial consisting of stepwise absorbable poly-glycolic acid and poly-(lactic-co-glycolic) acid and a supportive fine net of non-absorbable polyvinylidene fluoride. An initial boost of TGF-beta 1 or TGF-beta 1 and hyaluronan was applied and compared with controls. Matrix formation was assessed at days 7 and 21 by (1) histological staining of the typical extracellular matrix molecules proteoglycan and type I and type II collagens and by (2) real-time gene expression analysis of aggrecan, decorin, biglycan, type I, II, III, and X collagens as well as of catabolic matrix metalloproteinases MMP-2 and MMP-13. RESULTS: An initial boost with TGF-beta 1 or TGF-beta 1 and hyaluronan did not enhance the expression of characteristic AF matrix molecules in our 3D culture system. AF cells showed high viability in the progressively degrading biomaterial. Stratification by grade of intervertebral disc degeneration showed that AF cells from both, slightly degenerated, or severely degenerated tissue are capable of significant up-regulations of characteristic matrix molecules in 3D culture. AF cells from severely degenerated tissue, however, displayed significantly lower up-regulations in some matrix molecules such as aggrecan. CONCLUSIONS: We failed to show a supportive effect of an initial boost with TGF-beta 1 in our 3D culture system. This underlines the need for further investigations on growth factor releasing systems.

Human platelet-rich plasma induces chondrogenic differentiation of subchondral progenitor cells in polyglycolic acid-hyaluronan scaffolds.

Cartilage repair approaches may be improved by addition of human platelet-rich plasma (PRP) that increases chondrogenic differentiation of mesenchymal stem and progenitor cells. The aim of our study was to evaluate the effect of human PRP on the differentiation of multipotent human subchondral progenitor cells in resorbable polyglycolic acid-hyaluronan (PGA-HA) scaffolds. PGA-HA scaffolds were loaded with subchondral progenitor cells and stimulated with transforming growth factor-beta3 (TGFB3) or 5% PRP, whereas nonstimulated cultures served as controls. Chondrogenic differentiation was evaluated by real-time gene expression analysis of typical chondrogenic marker genes and by immunohistochemical staining of extracellular cartilage matrix molecules such as proteoglycans and collagen type II. TGFB3 and PRP induced the expression of chondrogenic marker genes collagen type II and IX, aggrecan, and cartilage oligomeric matrix protein in subchondral progenitor cells cultured in PGA-HA scaffolds compared with nonstimulated controls. Progenitor cells in PGA-HA scaffolds formed an extracellular matrix rich in proteoglycans and collagen type II on treatment with PRP, but to a lesser extent, than in cultures stimulated with TGFB3. The results suggest that PRP induces chondrogenic differentiation of progenitor cells in PGA-HA scaffolds and may be therefore beneficial in scaffold-assisted cartilage repair approaches involving stem and progenitor cells.

Regeneration of nucleus pulposus tissue in an ovine intervertebral disc degeneration model by cell-free resorbable polymer scaffolds.

Degeneration of intervertebral discs (IVDs) occurs frequently and is often associated with lower back pain. Recent treatment options are limited and treat the symptoms rather than regenerate the degenerated disc. Cell-free, freeze-dried resorbable polyglycolic acid (PGA)-hyaluronan implants were used in an ovine IVD degeneration model. The nucleus pulposus of the IVD was partially removed, endoscopically. PGA-hyaluronan implants were immersed in autologous sheep serum and implanted into the disc defect. Animals with nucleotomy only served as controls. The T2-weighted/fat suppression sequence signal intensity index of the operated discs, as assessed by magnetic resonance imaging (MRI), showed that implantation of the PGA-hyaluronan implant improved (p = 0.0066) the MRI signal compared to controls at 6 months after surgery. Histological analysis by haematoxylin and eosin and safranin O staining showed the ingrowth of cells with typical chondrocytic morphology, even cell distribution, and extracellular matrix rich in proteoglycan. Histomorphometric analyses confirmed that the implantation of the PGA-hyaluronan scaffolds improved (p = 0.027) the formation of regenerated tissue after nucleotomy. Disc heights remained stable in discs with nucleotomy only as well as after implantation of the implant. In conclusion, implantation of cell-free polymer-based implants after nucleotomy induces nucleus pulposus tissue regeneration and improves disc water content in the ovine model.

Polyglycolic acid-hyaluronan scaffolds loaded with bone marrow-derived mesenchymal stem cells show chondrogenic differentiation in vitro and cartilage repair in the rabbit model.

In cartilage repair, scaffold-assisted one-step approaches are used to improve the microfracture (Mfx) technique. Since the number of progenitors in Mfx is low and may further decrease with age, aim of our study was to analyze the chondrogenic potential of freeze-dried polyglycolic acid-hyaluronan (PGA-HA) implants preloaded with mesenchymal stem cells (MSCs) in vitro and in a rabbit articular cartilage defect model. Human bone marrow-derived MSC from iliac crest were cultured in freeze-dried PGA-HA implants for chondrogenic differentiation. In a pilot study, implants were loaded with autologous rabbit MSC and used to cover 5 mm × 6 mm full-thickness femoral articular cartilage defects (n = 4). Untreated defects (n = 3) served as controls. Gene expression analysis and histology showed induction of typical chondrogenic marker genes like type II collagen and formation of hyaline-like cartilaginous tissue in MSC-laden PGA-HA implants. Histological evaluation of rabbit repair tissue formation after 30 and 45 days showed formation of repair tissue, rich in chondrocytic cells and of a hyaline-like appearance. Controls showed no articular resurfacing, tissue repair in the subchondral zone and fibrin formation. These results suggest that MSC-laden PGA-HA scaffolds have chondrogenic potential and are a promising option for stem cell-mediated cartilage regeneration.