A Histological Analysis and Detection of Complement Regulatory Protein CD55 in SARS-CoV-2 Infected Lungs.

BACKGROUND: A complement imbalance in lung alveolar tissue can play a deteriorating role in COVID-19, leading to acute respiratory distress syndrome (ARDS). CD55 is a transmembrane glycoprotein that inhibits the activation of the complement system at the intermediate cascade level, blocking the activity of the C3 convertase. OBJECTIVE: In our study, lung specimens from COVID-19 and ARDS-positive COVID+/ARDS+ patients were compared with COVID-19 and ARDS-negative COVID-/ARDS- as well as COVID-/ARDS+ patients. METHODS: Histochemical staining and immunolabeling of CD55 protein were performed. RESULTS: The COVID-/ARDS- specimen showed higher expression and homogeneous distribution of glycosaminoglycans as well as compactly arranged elastic and collagen fibers of the alveolar walls in comparison to ARDS-affected lungs. In addition, COVID-/ARDS- lung tissues revealed stronger and homogenously distributed CD55 expression on the alveolar walls in comparison to the disrupted COVID-/ARDS+ lung tissues. CONCLUSIONS: Even though the collapse of the alveolar linings and the accumulation of cellular components in the alveolar spaces were characteristic of COVID+/ARDS+ lung tissues, evaluating CD55 expression could be relevant to understand its relation to the disease. Furthermore, targeting CD55 upregulation as a potential therapy could be an option for post-infectious complications of COVID-19 and other inflammatory lung diseases in the future.

In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts.

Pulsed electromagnetic field stimulation (PEMF) is gaining more attention as a non-invasive arthritis treatment. In our study, immortalized synovial fibroblasts (K4IM) derived from a non-arthritic donor were exposed to MAGCELL® ARTHRO, a PEMF device, with 105 mT intensity, 8 Hz frequency, and 2 × 2.5 min sessions conducted thrice with a 1 h interval, to understand the underlying mechanism in regard to the complement system. Additionally, tumor necrosis factor (TNFα, 10 ng/mL) pre-treatment prior to PEMF stimulation, as well as 3-day versus 6-day stimulation, were compared. Gene expression of C4b binding protein-alpha and -beta (C4BPα, C4BPß), complement factor (CF)-H, CFI, CD55, CD59, Interleukin (IL-6) and TNFα was analyzed. Immunofluorescence staining of CD55, CD59, and Ki67 was conducted. Results showed the absence of C4BPα gene expression, but C4BPß was present. One and three days of PEMF stimulation caused no significant changes. However, after six days, there was a significant increase in CD55, CFH, and CD59 gene expression, indicating cytoprotective effects. Conversely, IL-6 gene expression increased after six days of stimulation and even after a single session in TNFα pre-stimulated cells, indicating a pro-inflammatory effect. PEMF's ambivalent, i.e., enhancing complement regulatory proteins and pro-inflammatory cytokines, highlights its complexity at the molecular level.

A New Immortalized Human Lacrimal Gland Cell Line.

The lacrimal gland is crucial for maintaining ocular health by producing the aqueous component of the tear film, which hydrates and nourishes the ocular surface. Decreased production of this component results in dry eye disease, a condition affecting over 250 million people worldwide. However, the scarcity of primary human material for studying its underlying mechanisms and the absence of a cell model for human lacrimal gland epithelial cells present significant challenges. Here, we describe the generation of immortalized human lacrimal gland cell lines through the introduction of an SV40 antigen. We successfully isolated and characterized three cell clones from a female lacrimal gland donor, confirming their epithelial identity through genomic and protein analyses, including PCR, RNAseq, immunofluorescence and cultivation in a 3D spheroid model. Our findings represent a significant advancement, providing improved accessibility to investigate the molecular pathogenesis mechanisms of dry eye disease and potential therapeutic interventions. We identified the expression of typical epithelial cell marker genes and demonstrated the cells' capability to form 2D cell sheets and 3D spheroids. This establishment of immortalized human lacrimal gland cells with epithelial characteristics holds promise for future comprehensive studies, contributing to a deeper understanding of dry eye disease and its cellular mechanisms.

The Phenotype of Mesenchymal Stromal Cell and Articular Chondrocyte Cocultures on Highly Porous Bilayer Poly-L-Lactic Acid Scaffolds Produced by Thermally Induced Phase Separation and Supplemented with Hydroxyapatite.

Bilayer scaffolds could provide a suitable topology for osteochondral defect repair mimicking cartilage and subchondral bone architecture. Hence, they could facilitate the chondro- and osteogenic lineage commitment of multipotent mesenchymal stromal cells (MSCs) with hydroxyapatite, the major inorganic component of bone, stimulating osteogenesis. Highly porous poly-L-lactic acid (PLLA) scaffolds with two layers of different pore sizes (100 and 250 µm) and hydroxyapatite (HA) supplementation were established by thermally induced phase separation (TIPS) to study growth and osteogenesis of human (h) MSCs. The topology of the scaffold prepared via TIPS was characterized using scanning electron microscopy (SEM), a microCT scan, pycnometry and gravimetric analysis. HMSCs and porcine articular chondrocytes (pACs) were seeded on the PLLA scaffolds without/with 5% HA for 1 and 7 days, and the cell attachment, survival, morphology, proliferation and gene expression of cartilage- and bone-related markers as well as sulfated glycosaminoglycan (sGAG) synthesis were monitored. All scaffold variants were cytocompatible, and hMSCs survived for the whole culture period. Cross-sections revealed living cells that also colonized inner scaffold areas, producing an extracellular matrix (ECM) containing sGAGs. The gene expression of cartilage and bone markers could be detected. HA represents a cytocompatible supplement in PLLA composite scaffolds intended for osteochondral defects.

Anaphylatoxins and their corresponding receptors as potential drivers in cartilage calcification during osteoarthritis progression.

OBJECTIVE: The complement cascade as major fluid phase innate immune system is activated during progression of osteoarthritis (OA). Generated anaphylatoxins and the corresponding receptors C3aR and C5aR1 are associated with the calcification of blood vessels and involved in osteogenic differentiation. This study aims on elucidating whether complement activation products contribute to cartilage calcification of OA cartilage. METHOD: Human articular chondrocytes were osteogenically differentiated in vitro in the presence or absence of C3a, C5a, and bone morphogenetic protein (BMP) 2. Furthermore, macroscopically intact (OARSI grade ≤ 1) and highly degenerated human cartilage (OARSI grade ≥ 3) was used for C3aR and C5aR1 histochemistry. Calcification of the cartilage was assessed by Alizarin Red S and von Kossa staining. RESULTS: C3a and C5a amplified matrix mineralization during in vitro osteogenesis, while inhibition of the corresponding receptors impaired calcium deposition. Moreover, C3aR and C5aR1 expression was upregulated during osteogenic differentiation and also in degenerated cartilage. Additionally, anaphylatoxin receptor expression was positively associated with calcification of native cartilage tissue and calcium deposition during osteogenic differentiation. Finally, the pro-hypertrophic growth factor BMP2 induced the expression of C5aR1. CONCLUSIONS: Our findings indicate that anaphylatoxins and their receptors play a decisive role in cartilage calcification processes during OA progression.

Infrapatellar Fat Pad Modulates Osteoarthritis-Associated Cytokine and MMP Expression in Human Articular Chondrocytes.

Osteoarthritis (OA) most frequently affects the knee joint and is associated with an elevated expression of cytokines and extracellular cartilage matrix (ECM), degrading enzymes such as matrix metalloproteinases (MMPs). Differences in gene expression of the intra-articularly located infrapatellar fat pad (IPFP) and other fatty tissue suggest its autonomous function, yet its role in OA pathogenesis remains unknown. Human IPFPs and articular cartilage were collected from OA patients undergoing total knee arthroplasty, and biopsies from the IPFP of healthy patients harvested during knee arthroscopy served as controls (CO). Isolated chondrocytes were co-cultured with either osteoarthritic (OA) or CO-IPFPs in a transwell system. Chondrocyte expression of MMP1, -3, -13, type 1 and 2 collagens, interleukin IL1ß, IL6, IL10, and tumor necrosis factor TNFα was analyzed by RTD-PCR at day 0 and day 2, and TNFα secretion was analyzed by ELISA. The cytokine release in IPFPs was assessed by an array. Results: Both IPFPs (CO, OA) significantly reduced the expression of type 2 collagen and TNFα in chondrocytes. On the other hand, only CO-IPFP suppressed the expression of type 1 collagen and significantly induced the MMP13 expression. On the contrary, IL1ß and IL6 were significantly induced when exposed to OA-IPFP. Conclusions: The partial loss of the suppressive effect on type 1 collagen gene expression found for OA-IPFP shows the pathological remodeling and dedifferentiation potential of the OA-IPFP on the chondrocytes. However, the significant suppression of TNFα implies that the OA- and CO-IPFP could also exhibit a protective role in the knee joint, preventing the progress of inflammation.

Could an Anterior Cruciate Ligament Be Tissue-Engineered from Silk?

Silk has a long history as an exclusive textile, but also as a suture thread in medicine; nowadays, diverse cell carriers are manufactured from silk. Its advantages are manifold, including high biocompatibility, biomechanical strength and processability (approved for nearly all manufacturing techniques). Silk's limitations, such as scarcity and batch to batch variations, are overcome by gene technology, which allows for the upscaled production of recombinant "designed" silk proteins. For processing thin fibroin filaments, the sericin component is generally removed (degumming). In contrast to many synthetic biomaterials, fibroin allows for superior cell adherence and growth. In addition, silk grafts demonstrate superior mechanical performance and long-term stability, making them attractive for anterior cruciate ligament (ACL) tissue engineering. Looking at these promising properties, this review focusses on the responses of cell types to silk variants, as well as their biomechanical properties, which are relevant for ACL tissue engineering. Meanwhile, sericin has also attracted increasing interest and has been proposed as a bioactive biomaterial with antimicrobial properties. But so far, fibroin was exclusively used for experimental ACL tissue engineering approaches, and fibroin from spider silk also seems not to have been applied. To improve the bone integration of ACL grafts, silk scaffolds with osteogenic functionalization, silk-based tunnel fillers and interference screws have been developed. Nevertheless, signaling pathways stimulated by silk components remain barely elucidated, but need to be considered during the development of optimized silk cell carriers for ACL tissue engineering.

Does Vitamin K2 Influence the Interplay between Diabetes Mellitus and Intervertebral Disc Degeneration in a Rat Model?

Intervertebral disc (IVD) degeneration is a common cause of low back pain in diabetes mellitus type 2 (T2DM) patients. Its pathogenesis and the vitamin (vit.) K2 influence on this disease remain unclear. Lumbar motion segments of male Zucker Diabetes Fatty (ZDF) rats (non-diabetic [control] and diabetic; fed without or with vit. K2) were used. Femur lengths and vertebral epiphyseal cross-section areas were measured. IVDs were histopathologically examined. Protein synthesis and gene expression of isolated IVD fibrochondrocytes were analyzed. T2DM rats showed histopathological IVD degeneration. Femur lengths and epiphyseal areas were smaller in T2DM rats regardless of vit. K2 feeding. Fibrochondrocytes synthesized interleukin (IL)-24 and IL-10 with no major differences between groups. Alpha smooth muscle actin (αSMA) was strongly expressed, especially in cells of vit. K2-treated animals. Gene expression of aggrecan was low, and that of collagen type 2 was high in IVD cells of diabetic animals, whether treated with vit. K2 or not. Suppressor of cytokine signaling (Socs)3 and heme oxygenase (Hmox)1 gene expression was highest in the cells of diabetic animals treated with vit. K2. Vit. K2 influenced the expression of some stress-associated markers in IVD cells of diabetic rats, but not that of IL-10 and IL-24.

Sample size considerations in soft tissue biomechanics.

Biomechanical experiments help link tissue morphology with load-deformation characteristics. A tissue-dependent minimum sample number is indispensable to obtain accurate material properties. Stress-strain properties were retrieved from human dura mater and scalp skin, exemplifying two distinct soft tissues. Minimum sample sizes necessary for a stable estimation of material properties were obtained in a simulation study. One-thousand random samples were sequentially drawn for calculating the point at which a majority of the estimators settled within a corridor of stability at given tolerance levels around a 'complete' reference for the mean, median and coefficient of variation. Stable estimations of means and medians can be achieved below sample sizes of 30 at a ± 20%-tolerance within 80%-conformity for scalp skin and dura. Lower tolerance levels or higher conformity dramatically increase the required sample size. Conformity was barely ever reached for the coefficient of variation. The parameter type appears decisive for achieving conformity. STATEMENT OF SIGNIFICANCE: Biomechanical trials utilizing human tissues are needed to obtain material properties for surgical repair, tissue engineering and modeling purposes. Linking tissue mechanics with morphology helps elucidate form-function relationships, the 'morpho-mechanical link'. For material properties to be accurate, it is vital to examine a minimum number of samples. This number may vary between tissues, and the effects of intrinsic tissue characteristics on data accuracy are unclear to date. This study used data obtained from human dura and skin to compute minimum sample sizes required for estimating material properties at a stable level. It was shown that stable estimations are possible at a ± 20%-tolerance within 80%-conformity below sample sizes of 30. Higher accuracy warrants much higher sample sizes for most material properties.

Anatomical Tissue Engineering of the Anterior Cruciate Ligament Entheses.

The firm integration of anterior cruciate ligament (ACL) grafts into bones remains the most demanding challenge in ACL reconstruction, since graft loosening means graft failure. For a functional-tissue-engineered ACL substitute to be realized in future, robust bone attachment sites (entheses) have to be re-established. The latter comprise four tissue compartments (ligament, non-calcified and calcified fibrocartilage, separated by the tidemark, bone) forming a histological and biomechanical gradient at the attachment interface between the ACL and bone. The ACL enthesis is surrounded by the synovium and exposed to the intra-articular micromilieu. This review will picture and explain the peculiarities of these synovioentheseal complexes at the femoral and tibial attachment sites based on published data. Using this, emerging tissue engineering (TE) strategies addressing them will be discussed. Several material composites (e.g., polycaprolactone and silk fibroin) and manufacturing techniques (e.g., three-dimensional-/bio-printing, electrospinning, braiding and embroidering) have been applied to create zonal cell carriers (bi- or triphasic scaffolds) mimicking the ACL enthesis tissue gradients with appropriate topological parameters for zones. Functionalized or bioactive materials (e.g., collagen, tricalcium phosphate, hydroxyapatite and bioactive glass (BG)) or growth factors (e.g., bone morphogenetic proteins [BMP]-2) have been integrated to achieve the zone-dependent differentiation of precursor cells. However, the ACL entheses comprise individual (loading history) asymmetric and polar histoarchitectures. They result from the unique biomechanical microenvironment of overlapping tensile, compressive and shear forces involved in enthesis formation, maturation and maintenance. This review should provide a road map of key parameters to be considered in future in ACL interface TE approaches.

The chemicals between us-First results of the cluster analyses on anatomy embalming procedures in the German-speaking countries.

Hands-on courses utilizing preserved human tissues for educational training offer an important pathway to acquire basic anatomical knowledge. Owing to the reevaluation of formaldehyde limits by the European Commission, a joint approach was chosen by the German-speaking anatomies in Europe (Germany, Austria, Switzerland) to find commonalities among embalming protocols and infrastructure. A survey comprising 537 items was circulated to all anatomies in German-speaking Europe. Clusters were established for "ethanol"-, formaldehyde-based ("FA"), and "other" embalming procedures, depending on the chemicals considered the most relevant for each protocol. The logistical framework, volumes of chemicals, and infrastructure were found to be highly diverse between the groups and protocols. Formaldehyde quantities deployed per annum were three-fold higher in the "FA" (223 L/a) compared to the "ethanol" (71.0 L/a) group, but not for "other" (97.8 L/a), though the volumes injected per body were similar. "FA" was strongly related to table-borne air ventilation and total fixative volumes ≤1000 L. "Ethanol" was strongly related to total fixative volumes >1000 L, ceiling- and floor-borne air ventilation, and explosion-proof facilities. Air ventilation was found to be installed symmetrically in the mortuary and dissection facilities. Certain predictors exist for the interplay between the embalming used in a given infrastructure and technical measures. The here-established cluster analysis may serve as decision supportive tool when considering altering embalming protocols or establishing joint protocols between institutions, following a best practice approach to cater toward best-suited tissue characteristics for educational purposes, while simultaneously addressing future demands on exposure limits.

Co-Culture of Mesenchymal Stem Cells and Ligamentocytes on Triphasic Embroidered Poly(L-lactide-co-ε-caprolactone) and Polylactic Acid Scaffolds for Anterior Cruciate Ligament Enthesis Tissue Engineering.

Successful anterior cruciate ligament (ACL) reconstructions strive for a firm bone-ligament integration. With the aim to establish an enthesis-like construct, embroidered functionalized scaffolds were colonized with spheroids of osteogenically differentiated human mesenchymal stem cells (hMSCs) and lapine (l) ACL fibroblasts in this study. These triphasic poly(L-lactide-co-ε-caprolactone) and polylactic acid (P(LA-CL)/PLA) scaffolds with a bone-, a fibrocartilage transition- and a ligament zone were colonized with spheroids directly after assembly (DC) or with 14-day pre-cultured lACL fibroblast and 14-day osteogenically differentiated hMSCs spheroids (=longer pre-cultivation, LC). The scaffolds with co-cultures were cultured for 14 days. Cell vitality, DNA and sulfated glycosaminoglycan (sGAG) contents were determined. The relative gene expressions of collagen types I and X, Mohawk, Tenascin C and runt-related protein (RUNX) 2 were analyzed. Compared to the lACL spheroids, those with hMSCs adhered more rapidly. Vimentin and collagen type I immunoreactivity were mainly detected in the hMSCs colonizing the bone zone. The DNA content was higher in the DC than in LC whereas the sGAG content was higher in LC. The gene expression of ECM components and transcription factors depended on cell type and pre-culturing condition. Zonal colonization of triphasic scaffolds using spheroids is possible, offering a novel approach for enthesis tissue engineering.

In vivo ligamentogenesis in embroidered poly(lactic-co-ε-caprolactone) / polylactic acid scaffolds functionalized by fluorination and hexamethylene diisocyanate cross-linked collagen foams.

Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-ε-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or F combined with the foam (F + coll). Cell-free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, cell vitality and content, histo(patho)logy of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed.Scaffolds did not affect mice weight development and organs, indicating no organ toxicity. Moreover, scaffolds maintained their size and shape and reflected a high cell viability prior to and following implantation. Coll or F + coll scaffolds seeded with cells yielded superior macroscopic properties compared to the controls. Mild signs of inflammation (foreign-body giant cells and hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable after explantation, elastic modulus, maximum force, tensile strength and strain at Fmax were significantly lower in explanted scaffolds compared to those before implantation, with no significant differences between scaffold subtypes, except for a higher maximum force in F + coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering. a Lapine anterior cruciate ligament (LACL): red arrow, posterior cruciate ligament: yellow arrow. Medial anterior meniscotibial ligament: black arrow. b Explant culture to isolate LACL fibroblasts. c Scaffold variants: co: controls; F: functionalization by gas-phase fluorination; coll: collagen foam cross-linked with hexamethylene diisocyanate (HMDI). c1-2 Embroidery pattern of the scaffolds. d Scaffolds were seeded with LACL fibroblasts using a dynamical culturing approach as depicted. e Scaffolds were implanted subnuchally into nude mice, fixed at the nuchal ligament and sacrospinal muscle tendons. f Two weeks after implantation. g Summary of analyses performed. Scale bars 1 cm (b, d), 0.5 cm (c). (sketches drawn by G.S.-T. using Krita 4.1.7 [Krita foundation, The Netherlands]).

Healing of Ligaments and Tendons: Tissue Engineering and Models.

The aim of this Special Issue is to summarize the latest developments in tendon/ligament research and tissue engineering (TE), providing helpful approaches for future tendon/ligament reconstruction (Figure 1) [...].

Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα.

Background: Case reports are available showing that patients develop symptoms of acute arthritis during or after recovery from SARS-CoV-2 infection. Since the interrelation is still unknown, our aim was to study the impact of the SARS-CoV-2 nucleocapsid protein (NP) on human fibroblast-like synoviocytes and human endothelial cells (hEC) in terms of complement and cytokine regulation. Methods: Non-arthritic (K4IM) synoviocyte, arthritic (HSE) synoviocyte cell lines and primary hEC were stimulated with recombinant NP and/or TNFα. Analyses of cell viability, proliferation, gene and protein expression of cytokines and complement factors were performed. Results: NP suppressed significantly the vitality of hEC and proliferation of HSE. NP alone did not induce any significant changes in the examined gene expressions. However, NP combined with TNFα induced significantly higher TNFα in HSE and K4IM as well as higher IL-6 and CD55 gene expression in HSE and suppressed C3aR1 gene expression in hEC. HSE proliferated twice as fast as K4IM, but showed significantly lesser gene expressions of CD46, CD55, CD59 and TNFα with significantly higher IL-6 gene expression. CD35 gene expression was undetectable in K4IM, HSE and hEC. Conclusions: NP might contribute in combination with other inflammatory factors to complement regulation in arthritis.

Tendon healing: a concise review on cellular and molecular mechanisms with a particular focus on the Achilles tendon.

Tendon is a bradytrophic and hypovascular tissue, hence, healing remains a major challenge. The molecular key events involved in successful repair have to be unravelled to develop novel strategies that reduce the risk of unfavourable outcomes such as non-healing, adhesion formation, and scarring. This review will consider the diverse pathophysiological features of tendon-derived cells that lead to failed healing, including misrouted differentiation (e.g. de- or transdifferentiation) and premature cell senescence, as well as the loss of functional progenitors. Many of these features can be attributed to disturbed cell-extracellular matrix (ECM) or unbalanced soluble mediators involving not only resident tendon cells, but also the cross-talk with immigrating immune cell populations. Unrestrained post-traumatic inflammation could hinder successful healing. Pro-angiogenic mediators trigger hypervascularization and lead to persistence of an immature repair tissue, which does not provide sufficient mechano-competence. Tendon repair tissue needs to achieve an ECM composition, structure, strength, and stiffness that resembles the undamaged highly hierarchically ordered tendon ECM. Adequate mechano-sensation and -transduction by tendon cells orchestrate ECM synthesis, stabilization by cross-linking, and remodelling as a prerequisite for the adaptation to the increased mechanical challenges during healing. Lastly, this review will discuss, from the cell biological point of view, possible optimization strategies for augmenting Achilles tendon (AT) healing outcomes, including adapted mechanostimulation and novel approaches by restraining neoangiogenesis, modifying stem cell niche parameters, tissue engineering, the modulation of the inflammatory cells, and the application of stimulatory factors.Cite this article: Bone Joint Res 2022;11(8):561-574.

In vitro evaluation of a synthetic (Biobrane®) and a biopolymer (Epicite) wound dressing with primary human juvenile and adult fibroblasts after different colonization strategies.

BACKGROUND: The three-dimensional [3D] wound dressings Biobrane® and Epicite are used in the wound management. Fibroblasts are important for successful deep wound healing. The direct effect of Biobrane® and Epicite on human fibroblasts, particularly of juvenile individuals, remains unclear. Therefore, this study compared the survival and growth characteristics of juvenile and adult dermal fibroblasts on Biobrane® and Epicite using different culture models. METHOD: Murine (L929), primary juvenile and adult human fibroblasts were seeded on both materials using two dimensional (2D, slide culture) or 3D culture at the medium-air interface and dynamical rotatory culture. Cell adherence, viability, morphology, actin cytoskeleton architecture and DNA content were monitored. Scanning electron microscopy (SEM) analyses could be only performed from Biobrane®. Permeability of both materials were tested. RESULTS: The majority of all tested fibroblasts species survived on both dressings with no significant differences between 1 and 14 days. Juvenile and adult fibroblasts exerted typical fibroblast morphology with spindle-shaped cell bodies on the materials. SEM visualized morphological differences between murine and human fibroblasts on Biobrane®. Juvenile and adult fibroblasts colonized Biobrane® in rotatory culture after 7 days the most. The Biobrane® rotatory culture of L929 and juvenile fibroblasts showed after 7 days the significantly highest DNA amount. No major gender differences could be observed. Biobrane® had a higher permeability than Epicite. CONCLUSION: Both wound dressing can be colonized by fibroblasts suggesting their high cytocompatibility. Fibroblast survival and morphology on Biobrane® and Epicite depended on the culture system and the fibroblast source.

Biodegradable Poly(D-L-lactide-co-glycolide) (PLGA)-Infiltrated Bioactive Glass (CAR12N) Scaffolds Maintain Mesenchymal Stem Cell Chondrogenesis for Cartilage Tissue Engineering.

Regeneration of articular cartilage remains challenging. The aim of this study was to increase the stability of pure bioactive glass (BG) scaffolds by means of solvent phase polymer infiltration and to maintain cell adherence on the glass struts. Therefore, BG scaffolds either pure or enhanced with three different amounts of poly(D-L-lactide-co-glycolide) (PLGA) were characterized in detail. Scaffolds were seeded with primary porcine articular chondrocytes (pACs) and human mesenchymal stem cells (hMSCs) in a dynamic long-term culture (35 days). Light microscopy evaluations showed that PLGA was detectable in every region of the scaffold. Porosity was greater than 70%. The biomechanical stability was increased by polymer infiltration. PLGA infiltration did not result in a decrease in viability of both cell types, but increased DNA and sulfated glycosaminoglycan (sGAG) contents of hMSCs-colonized scaffolds. Successful chondrogenesis of hMSC-colonized scaffolds was demonstrated by immunocytochemical staining of collagen type II, cartilage proteoglycans and the transcription factor SOX9. PLGA-infiltrated scaffolds showed a higher relative expression of cartilage related genes not only of pAC-, but also of hMSC-colonized scaffolds in comparison to the pure BG. Based on the novel data, our recommendation is BG scaffolds with single infiltrated PLGA for cartilage tissue engineering.

Complement Proteins C5/C5a, Cathepsin D and Prolactin in Chondrocytes: A Possible Crosstalk in the Pathogenesis of Osteoarthritis.

INTRODUCTION: Both increased activity of the complement system (CS) and the role of the pituitary hormone prolactin (PRL) are implicated in osteoarthritis (OA) pathogenesis. Besides, Cathepsin D (CatD) activity is increased in the context of OA and can exert not only proteolytic but also non-proteolytic effects on cells. For the first time, possible crosstalk between two separate humoral systems: the CS and the PRL hormone systems in chondrocytes are examined together. METHODS: Primary human articular chondrocytes (hAC) were stimulated with complement protein C5 (10 µg /mL), PRL (25 ng/mL), CatD (100 ng/mL), or anaphylatoxin C5a (25 ng/mL) for 24 h or 72 h, while unstimulated cells served as controls. In addition, co-stimulations of C5 or PRL with CatD were carried out under the same conditions. The influence of the stimulants on cell viability, cell proliferation, and metabolic activity of hAC, the chondrosarcoma cell line OUMS-27, and endothelial cells of the human umbilical cord vein (HUVEC) was investigated. Gene expression analysis of C5a receptor (C5aR1), C5, complement regulatory protein CD59, PRL, PRL receptor (PRLR), CatD, and matrix metal-loproteinases (MMP)-13 were performed using real-time PCR. Also, collagen type (Col) I, Col II, C5aR1, CD59, and PRL were detected on protein level using immunofluorescence labeling. RESULTS: The stimulation of the hAC showed no significant impairment of the cell viability. C5, C5a, and PRL induced cell growth in OUMS-27 and HUVEC, but not in chondrocytes. CatD, as well as C5, significantly reduced the gene expression of CatD, C5aR1, C5, and CD59. PRLR gene expression was likewise impaired by C5, C5a, and PRL+CatD stimulation. On the protein level, CatD, as well as C5a, decreased Col II as well as C5aR1 synthesis. CONCLUSIONS: The significant suppression of the C5 gene expression under the influence of PRL+CatD and that of CD59 via PRL+/-CatD and conversely a suppression of the PRLR gene expression via C5 alone or C5a stimulation indicates an interrelation between the two mentioned systems. In addition, CatD and C5, in contrast to PRL, directly mediate possible negative feedback of their own gene expression.

Highly porous novel chondro-instructive bioactive glass scaffolds tailored for cartilage tissue engineering.

Cartilage injuries remain challenging since the regenerative capacity of cartilage is extremely low. The aim was to design a novel type of bioactive glass (BG) scaffold with suitable topology that allows the formation of cartilage-specific extracellular matrix (ECM) after colonization with chondrogenic cells for cartilage repair. Highly porous scaffolds with interconnecting pores consisting of 100 % BG were manufactured using a melting, milling, sintering and leaching technique. Scaffolds were colonized with porcine articular chondrocytes (pAC) and undifferentiated human mesenchymal stromal cells (hMSC) for up to 35 days. Scaffolds displayed high cytocompatibility with no major pH shift. Scanning electron microscopy revealed the intimate pAC-scaffold interaction with typical cell morphology. After 14 days MSCs formed cell clusters but still expressed cartilage markers. Both cell types showed aggrecan, SOX9 gene and protein expression, cartilage proteoglycan and sulfated glycosaminoglycan synthesis for the whole culture time. Despite type II collagen gene expression could not anymore be detected at day 35, protein synthesis was visualized for both cell types during the whole culturing period, increasing in pAC and declining after day 14 in hMSC cultures. The novel BG scaffold was stable, cytocompatible and cartilage-specific protein synthesis indicated maintenance of pAC's differentiated phenotype and chondro-instructive effects on hMSCs.