Influence of the Transmucosal Surface of Dental Implants on the Soft Tissue Attachment Level and Marginal Bone Loss in Preclinical Studies: A Systematic Review.

PURPOSE: To determine the characteristics of dental implant transmucosal surfaces that influence soft tissue attachment and marginal bone loss (MBL). MATERIALS AND METHODS: The PubMed, Embase, and Cochrane Library electronic databases were searched based on predefined PICO eligibility criteria. Data from animal studies that compared junctional epithelium and connective tissue attachment and MBL from 4 days to 72 weeks were analyzed. The risk of bias was performed with the Systematic Review Centre for Laboratory Animal Experimentation tool. A rank analysis evaluation of data was performed, and the most frequently appearing materials/surfaces for each tissue compartment were identified. RESULTS: The search identified 3,549 studies, 28 of which were eligible for analysis, with an average risk of bias of 28% ± 10%. Machined, polished, etched, sandblasted, or coated titanium and zirconia materials/surfaces were most frequently examined. Several studies investigated lithium disilicate, polyether ether ketone (PEEK) or polyether ketone ketone (PEKK), aluminum oxide, and gold. Based on ranking and frequency of use at different time points, titanium grade IV (Ti-4) microthreads with a polished neck area most frequently supported natural tooth-like junctional epithelial attachment (≤ 1.5 mm), while machined Ti-4 and machined titanium grade V (Ti-5) most frequently supported connective tissue attachment (≤ 1.25 mm) and led to the least MBL (≤ 0.75 mm). CONCLUSIONS: Analyzed data suggest that Ti-4 microthreads with a polished neck area and machined Ti-4 and Ti-5 were the materials/surfaces of choice for the transmucosal part of implants. However, the extensive heterogeneity in reported studies precludes solid identification of the best materials/surfaces.

FastSkin® Concept: A Novel Treatment for Complex Acute and Chronic Wound Management.

Successful treatments for acute and chronic skin wounds remain challenging. The goal of this proof-of-concept study was to assess the technical feasibility and safety of a novel wound treatment solution, FastSkin®, in a pig model. FastSkin® was prepared from skin micrografts patterned in blood using acoustic waves. Upon coagulation, the graft was transferred on a silicone sheet and placed on wounds. Six full-thickness wounds were created at the back of two pigs and treated with either FastSkin®, split-thickness skin graft (positive control), a gauze coverage (negative control, NC1), or blood patterned without micrografts (negative control, NC2). Silicone sheets were removed after 7, 14, and 21 days. Wound healing was monitored for six weeks and evaluated macroscopically for re-epithelialization and morphometrically for residual wound area and wound contraction. Tissue regeneration was assessed with histology after six weeks. Re-epithelialization was faster in wounds covered with FastSkin® treatments compared to NC2 and in NC2 compared to NC1. Importantly, an enhanced collagen organization was observed in FastSkin® in contrast to NC treatments. In summary, two clinically approved skin wound treatments, namely micrografting and blood clot graft, were successfully merged with sound-induced patterning of micrografts to produce an autologous, simple, and biologically active wound treatment concept.

Biological Performance of Duplex PEO + CNT/PCL Coating on AZ31B Mg Alloy for Orthopedic and Dental Applications.

To regulate the degradation rate and improve the surface biocompatibility of the AZ31B magnesium alloy, three different coating systems were produced via plasma electrolytic oxidation (PEO): simple PEO, PEO incorporating multi-walled carbon nanotubes (PEO + CNT), and a duplex coating that included a polycaprolactone top layer (PEO + CNT/PCL). Surfaces were characterized by chemical content, roughness, topography, and wettability. Biological properties analysis included cell metabolism and adhesion. PEO ± CNT resulted in an augmented surface roughness compared with the base material (BM), while PCL deposition produced the smoothest surface. All surfaces had a contact angle below 90°. The exposure of gFib-TERT and bmMSC to culture media collected after 3 or 24 h did not affect their metabolism. A decrease in metabolic activity of 9% and 14% for bmMSC and of 14% and 29% for gFib-TERT was observed after 3 and 7 days, respectively. All cells died after 7 days of exposure to BM and after 15 days of exposure to coated surfaces. Saos-2 and gFib-TERT adhered poorly to BM, in contrast to bmMSC. All cells on PEO anchored into the pores with filopodia, exhibited tiny adhesion protrusions on PEO + CNT, and presented a web-like spreading with lamellipodia on PEO + CNT/PCL. The smooth and homogenous surface of the duplex PEO + CNT/PCL coating decreased magnesium corrosion and led to better biological functionality.

Design of customized soft tissue substitutes for anterior single-tooth and posterior double-tooth defects: An in vitro study.

OBJECTIVE: This study aims to validate the standardized procedure for designing soft tissue substitutes (STS) adapted to optimally fit single-tooth defects in the anterior jaws and double-tooth defects in the posterior jaw and to compare mathematically modeled average shapes. MATERIALS AND METHODS: Casts from 35 patients with 17 single-tooth defects in anterior region and 21 double-tooth defects in posterior region were scanned. STS were designed and sectioned in 3D slices meshes. Thickness values were documented respecting mesial-distal and buccal-lingual orientations. Graphs were embedded into images, and hierarchical clustering was applied to group STS according to shape and thickness. RESULTS: STS clustered into two groups per defect type. For anterior single defects, STS (n = 4) were either a small and thin oval: 7 mm buccal-lingual, 4-5 mm mesial-distal direction and 1.1-1.5 mm thick or a larger oval (n = 13): 9 mm buccal-lingual, 5-7 mm mesial-distal and 1.6 m thick. For posterior double tooth defects, STS (n = 10) were either narrow, long and thick: 6-7 mm buccal-lingual, 16-20 mm mesial-distal and 2.2 thick or a wide, thinner rectangle (n = 11): 9-11 mm buccal-lingual, 12-14 mm mesial-distal and 1.1-1.5 mm thick. CONCLUSIONS: The study validated the standardized digital method to design grafts for soft tissue volume augmentation and identified four average shapes for anterior single-tooth and posterior double-tooth soft tissue defects. CLINICAL SIGNIFICANCE: We developed and validated a standardized digital method to design an optimal geometrical shape of a soft tissue substitute for oral volume augmentation and combined it with mathematical modeling to identify average shapes for single-interior, and double-posterior tooth defects. The identified average shapes offer the possibility to produce better-fitted xenografts or synthetic STS blocks requiring minimal chair-side adaptation leading to reduced clinical time and patient discomfort and potentially improving soft tissue volume augmentation outcomes.

Influence of the Titanium Base Abutment Design on Monolithic Zirconia Multiple-Unit Implant Fixed Dental Prostheses: A Laboratory Study.

PURPOSE: This in vitro study evaluated technical outcomes of monolithic zirconia implant-supported fixed dental prostheses (iFDPs) supported by different designs of titanium base abutments after aging and static load testing. MATERIALS AND METHODS: Sixty three-unit monolithic zirconia (yttrium oxide partially stabilized tetragonal zirconia polycrystals; Y-TZP)iFDPs were produced and divided into four groups: group A-conical titanium base abutments for the prosthesis; group B-cylindrical titanium base abutments for the crown; group C-conical titanium base abutment for the prosthesis and cylindrical titanium base abutment for the crown; group D-cylindrical titanium base abutments for the prosthesis. The samples were subjected to an aging protocol and to continuous static loading until failure and analyzed visually and with specific software. The technical outcomes comprised the occurrence of debonding, screw loosening, deformation and fracture, abutment deformation and fracture, implant deformation and fracture, zirconia chipping and fracture, and bending moments. The Pearson chi-squared test (χ2) and Fischer exact test were used to compare the outcomes. The Kolmogorov-Smirnov test was used to evaluate data distribution of the bending moments. Analysis of variance (ANOVA) was used for the analysis of parametric data distribution, and the Kruskal-Wallis test was used for nonparametric data distribution. RESULTS: After aging, a higher percentage of debonding (P = .042) and micromovement (P = .034) was recorded in group C (P = .042). The conical titanium base abutments had a higher debonding (P = .049) and a higher macromovement rate (P = .05). The static load test showed higher bending moments in group D (P = .001) and lower bending moments in group A (P = .001). Debonding was highest in group C (P = .001) and lowest in group A (P = .002). Complete loss of retention rate was highest in group C (P = .001). The conical titanium base abutment had the highest debonding rate (P = .001) and complete loss of retention (P = .001). The micromovement rate was the highest for cylindrical titanium base abutments for the crown (P = .001). The lowest screw loosening, zirconia chipping, and fracture rate (P = .001) and the highest screw deformation (P = .004) were recorded in group A. The screw deformation rate was lowest in the cylindrical titanium base abutments for the crown (P = .008). CONCLUSION: The mixed titanium base abutment design comprising one conical and one cylindrical abutment in Y-TZP iFDPs led to the highest debonding rate. The cylindrical titanium base abutment for the prosthesis provided a lower percentage of debonding and the highest load resistance.

Design of customized soft-tissue substitutes for posterior single-tooth defects: A proof-of-concept in-vitro study.

OBJECTIVES: Soft-tissue volume augmentation treatments do not provide the satisfactory long-term functional and esthetic outcomes. The aim of the study was to develop a standardized digital procedure to design individual soft-tissue substitutes (STS) and apply mathematical modeling to obtain average shape STS for single posterior tooth defects. MATERIAL AND METHODS: Thirty-three casts from 30 patients were scanned. STS were designed with a computer-aided design software and a systematic procedure standardized the measurements across all STS using 3D-analysis software. The occlusal, mesial-distal, and buccal-lingual planes were defined to partition, each STS and produce a mesh. The thickness values of each 3D slice were documented in a coordinate system chart to generate a scatter graph. Graphs were embedded into images (Orange software) and images were analyzed via hierarchical clustering. RESULTS: Three STS groups were identified according to shape. Two shapes corresponded to the maxilla defects: a square (n = 13) with dimensions of 10 mm in a lingual-buccal (length) and 7-10 mm in a mesial-distal (width) direction; a rectangle (n = 11) of 11 mm in length and 4-7 mm in width. The average shape for mandible defects (n = 9) was smaller (6-8 mm in length, 5-10 mm in width). The highest thickness in all STS was in the buccal portion, above the alveolar ridge, with median values of 2 mm. The lowest thickness of 0.2 mm was at the edges. CONCLUSIONS: The study developed novel methodology to design customized, as well as average shape STS for volume augmentation. Future STS harboring adapted geometry might increase volume augmentation efficiency and accuracy, while reducing surgical time.

Quantitative measures of bone shape, cartilage morphometry and joint alignment are associated with disease in an ACLT and MMx rat model of osteoarthritis.

Multi-scale, subject-specific quantitative methods to characterize and monitor osteoarthritis in animal models and therapeutic treatments could help reveal causal relationships in disease development and distinguish treatment strategies. In this work, we demonstrate a reproducible and sensitive quantitative image analysis to characterize bone, cartilage and joint measures describing a rat model of post-traumatic osteoarthritis. Eleven 3-month-old male Wistar rats underwent medial anterior cruciate ligament (ACL) transection and medial meniscectomy on the right knee to destabilise the right tibiofemoral joint. They were sacrificed 6 weeks post-surgery and a silicon-based micro-bead contrast agent was injected in the joint space, before scanning with micro-computed tomography (microCT). Subsequently, 3D quantitative morphometric analysis (QMA), previously developed for rabbit joints, was performed. This included cartilage, subchondral cortical and epiphyseal bone measures, as well as novel tibiofemoral joint metrics. Semi-quantitative evaluation was performed on matching two-dimensional (2D) histology and microCT images. Reproducibility of the QMA was tested on eleven age-matched additional joints. The results indicate the QMA method is accurate and reproducible and that microCT-derived cartilage measurements are valid for the analysis of rat joints. The pathologic changes caused by transection of the ACL and medial meniscectomy were reflected in measurements of bone shape, cartilage morphology, and joint alignment. Furthermore, we were able to identify model-specific predictive parameters based on morphometric parameters measured with the QMA.

Human gingival fibroblast proliferation on materials used for dental implant abutments: a systematic review.

PURPOSE: To conduct a systematic review to evaluate the influence of materials and surfaces used for dental implant abutments on the proliferation of human gingival fibroblasts. MATERIALS AND METHODS: The focus question of this review was: Which material/surface characteristics used for dental implant abutments influence/enhance proliferation of human gingival fibroblasts? The Medline/PubMed, Embase, and Cochrane Library databases were searched using "gingiva," "fibroblasts," "proliferation," and "dental implant abutments" as main keywords with AND/OR as Boolean operators. In vitro studies reporting 3 to 4 or 6 to 7 days of cell proliferation, surface hydrophilicity, and roughness were included. A quality assessment of the selected studies was performed using the web-based Science in Risk Assessment and Policy (SciRAP) tool. RESULTS: The search identified 1,144 studies, and 44 were eligible for inclusion. The average reporting quality SciRAP score was 82.87 ± 10.68, and the average methodologic quality SciRAP score was 87.35 ± 10.55. Machined, polished, and coated titanium and zirconia surfaces were most commonly investigated. Several studies analyzed aluminum oxide, cobalt-chrome-molybdenum alloy, lithium disilicate, polyether ether ketone, polymer-infiltrated ceramic network, and bioglass. The best cell proliferation was observed on zirconia and on titanium harboring nanotubules or microgrooves. UV treatment, polydopamine, and nitride coatings also improved cell proliferation. Due to the heterogeneity of the data, no correlation could be established between cell profileration and surface hydrophilicity or roughness. However, surface roughness in the range of Ra = 15 to 145 nm and Sa = 19 to 500 nm on titanium and zirconia proved most suitable. CONCLUSION: Titanium surfaces with directional guidance patterning and zirconia surfaces best supported cell proliferation during the first week of cell culture. Lack of standardization in surface definitions (machined or polished), methodology, and reporting prevented analytical comparison and should be imposed in future studies.

3D Printing Approach in Dentistry: The Future for Personalized Oral Soft Tissue Regeneration.

Three-dimensional (3D) printing technology allows the production of an individualized 3D object based on a material of choice, a specific computer-aided design and precise manufacturing. Developments in digital technology, smart biomaterials and advanced cell culturing, combined with 3D printing, provide promising grounds for patient-tailored treatments. In dentistry, the "digital workflow" comprising intraoral scanning for data acquisition, object design and 3D printing, is already in use for manufacturing of surgical guides, dental models and reconstructions. 3D printing, however, remains un-investigated for oral mucosa/gingiva. This scoping literature review provides an overview of the 3D printing technology and its applications in regenerative medicine to then describe 3D printing in dentistry for the production of surgical guides, educational models and the biological reconstructions of periodontal tissues from laboratory to a clinical case. The biomaterials suitable for oral soft tissues printing are outlined. The current treatments and their limitations for oral soft tissue regeneration are presented, including "off the shelf" products and the blood concentrate (PRF). Finally, tissue engineered gingival equivalents are described as the basis for future 3D-printed oral soft tissue constructs. The existing knowledge exploring different approaches could be applied to produce patient-tailored 3D-printed oral soft tissue graft with an appropriate inner architecture and outer shape, leading to a functional as well as aesthetically satisfying outcome.

S100B + A1 CELISA: A Novel Potency Assay and Screening Tool for Redifferentiation Stimuli of Human Articular Chondrocytes.

During monolayer expansion, a necessary step in autologous chondrocyte implantation, human articular chondrocytes (HAC) dedifferentiate and lose their capacity to produce stable hyaline cartilage. Determining HAC potency and learning how to trigger their redifferentiation would improve cell-based cartilage regeneration therapies. We previously identified S100B and S100A1 proteins as markers of HAC redifferentiation potential. Here, we aimed to: (i) demonstrate a correlation between S100B + A1-positive HAC in monolayer culture and their neochondrogenesis capacity in pellet culture; (ii) develop an S100B + A1 cell-based ELISA, and (iii) prove that S100B + A1 induction in HAC increases their chondrogenic capacity. Expression patterns of S100A1 and S100B were investigated in HAC during dedifferentiation (monolayer) or redifferentiation (pellet or high-osmolarity/BMP4 treatment in monolayer) using qRT-PCR, immunocytochemistry, or immunohistochemistry. A cell-based ELISA (CELISA) was developed as a 96-well microplate multiplex assay to measure S100B + A1 (chondrogenesis), alkaline phosphatase (hypertrophy), and DNA amount (normalization), and applied to HAC, bone marrow-derived mesenchymal stem cells and the chondrocytic cell line ATDC5. The direct correlation between the percentage of S100B + A1-positive HAC in monolayer and their neochondrogenesis in pellets validates S100B + A1 as a marker of chondrogenic potency. The S100B + A1-CELISA accurately determines HAC differentiation status, allows identification of chondrogenic stimuli, and permits the simultaneous monitoring of the undesirable hypertrophic phenotype. This novel assay offers a high-throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and for identifying redifferentiation factors/conditions. HAC improved neochondrogenesis in pellets-induced with high-osmolarity and BMP4 treatment in monolayer-suggests that cell instruction prior to implantation may improve cartilage repair. J. Cell. Physiol. 232: 1559-1570, 2017. © 2016 Wiley Periodicals, Inc.

S100A1 and S100B: Calcium Sensors at the Cross-Roads of Multiple Chondrogenic Pathways.

The S100 protein family comprises more than 20 members of small calcium binding proteins operating as Ca2 +-activated switches that interact and modulate the activity of a large number of targets. S100A1 and S100B, two members of this family, have been recently associated with the differentiation status of human articular chondrocytes. Both proteins are homogeneously expressed in all cartilage zones, their expression decreases during chondrocyte dedifferentiation, and can be induced under conditions promoting redifferentiation. Although S100 proteins have a broad range of extra- and intracellular roles, functional studies of S100 proteins expressed in chondrocytes have focused on their extracellular roles linked to catabolic processes. The intracellular roles of S100A1 and S100B in chondrocytes remain largely unexplored, yet the few studies addressing their intracellular activity point toward potentially important functions in chondrocyte biology. This review summarizes reported intracellular S100A1 and S100B regulatory functions described in other cell types that could be also involved in the regulation of chondrogenic processes in cartilage. Potential roles of S100A1 and S100B in the TGF-ß-SMAD, the cAMP-PKA-CREB, and the PI3K-AKT pathways, Ca2+ homeostasis, cytoskeleton dynamics, the calcineurin-NFAT pathway, interactions with the p53 family, and the Hippo pathway are examined in the context of chondrocyte biology. Based on the plethora of interactions of S100A1 and S100B with different molecular partners playing essential roles in chondrocyte biology, and the staggering complexity and ubiquity of cross-talk among these partners, we hypothesize that these S100 proteins play fundamental roles in the spatial and temporal regulation of chondrogenesis. J. Cell. Physiol. 232: 1979-1987, 2017. © 2016 Wiley Periodicals, Inc.

Three-Dimensional Quantitative Morphometric Analysis (QMA) for In Situ Joint and Tissue Assessment of Osteoarthritis in a Preclinical Rabbit Disease Model.

This work utilises advances in multi-tissue imaging, and incorporates new metrics which define in situ joint changes and individual tissue changes in osteoarthritis (OA). The aims are to (1) demonstrate a protocol for processing intact animal joints for microCT to visualise relevant joint, bone and cartilage structures for understanding OA in a preclinical rabbit model, and (2) introduce a comprehensive three-dimensional (3D) quantitative morphometric analysis (QMA), including an assessment of reproducibility. Sixteen rabbit joints with and without transection of the anterior cruciate ligament were scanned with microCT and contrast agents, and processed for histology. Semi-quantitative evaluation was performed on matching two-dimensional (2D) histology and microCT images. Subsequently, 3D QMA was performed; including measures of cartilage, subchondral cortical and epiphyseal bone, and novel tibio-femoral joint metrics. Reproducibility of the QMA was tested on seven additional joints. A significant correlation was observed in cartilage thickness from matching histology-microCT pairs. The lateral compartment of operated joints had larger joint space width, thicker femoral cartilage and reduced bone volume, while osteophytes could be detected quantitatively. Measures between the in situ tibia and femur indicated an altered loading scenario. High measurement reproducibility was observed for all new parameters; with ICC ranging from 0.754 to 0.998. In conclusion, this study provides a novel 3D QMA to quantify macro and micro tissue measures in the joint of a rabbit OA model. New metrics were established consisting of: an angle to quantitatively measure osteophytes (σ), an angle to indicate erosion between the lateral and medial femoral condyles (ρ), a vector defining altered angulation (λ, α, ß, γ) and a twist angle (τ) measuring instability and tissue degeneration between the femur and tibia, a length measure of joint space width (JSW), and a slope and intercept (m, Χ) of joint contact to demonstrate altered loading with disease progression, as well as traditional bone and cartilage and histo-morphometry measures. We demonstrate correlation of microCT and histology, sensitive discrimination of OA change and robust reproducibility.

The Role of Cells in Meniscal Guided Tissue Regeneration: A Proof of Concept Study in a Goat Model.

OBJECTIVE: Successful repair of defects in the avascular zone of meniscus remains a challenge in orthopedics. This proof of concept study aimed to investigate a guided tissue regeneration approach for treatment of tears in meniscus avascular zone in a goat model. DESIGN: Full-depth longitudinal tear was created in the avascular zone of the meniscus and sutured. In the two treatment groups, porcine collagen membrane was wrapped around the tear without (CM) or with injection of expanded autologous chondrocytes (CM+cells), whereas in the control group the tear remained only sutured. Gait recovery was evaluated during the entire follow-up period. On explantation at 3 and 6 months, macroscopic gross inspection assessed healing of tears, degradation of collagen membrane, potential signs of inflammation, and osteoarthritic changes. Microscopic histology scoring criteria were developed to evaluate healing of tears, the cellular response, and the inflammatory response. RESULTS: Gait recovery suggested protective effect of collagen membrane and was supported by macroscopical evaluation where improved tear healing was noted in both treated groups. Histology scoring in CM compared to suture group revealed an increase in tear margins contact, newly formed connective tissue between margins, and cell formations surrounded with new matrix after 3 months yet not maintained after 6 months. In contrast, in the CM+cells group these features were observed after 3 and 6 months. CONCLUSIONS: A transient, short-term guided tissue regeneration of avascular meniscal tears occurred upon application of collagen membrane, whereas addition of expanded autologous chondrocytes supported more sustainable longer term tear healing.

Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro.

BACKGROUND: Platelet-rich concentrates are used as a source of growth factors to improve the healing process. The diverse preparation protocols and the gaps in knowledge of their biological properties complicate the interpretation of clinical results. QUESTIONS/PURPOSES: In this study we aimed to (1) analyze the concentration and kinetics of growth factors released from leukocyte- and platelet-rich fibrin (L-PRF), leukocyte- and platelet-rich plasma (L-PRP), and natural blood clot during in vitro culture; (2) investigate the migration of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as a functional response to the factors released; and (3) uncover correlations between individual growth factors with the initial platelet/leukocyte counts or the induced cell migration. METHODS: L-PRF, L-PRP, and natural blood clot prepared from 11 donors were cultured in vitro for 28 days and media supernatants collected after 8 hours and 1, 3, 7, 14, and 28 days. Released transforming growth factor ß1 (TGF-ß1), vascular endothelial growth factor (VEGF), insulin growth factor (IGF-1), platelet-derived growth factor AB (PDGF-AB), and interleukin-1ß (IL-1ß) were measured in the supernatants with enzyme-linked immunosorbent assay. Migration of MSC and HUVEC induced by the supernatants was evaluated in Boyden chambers. RESULTS: More TGF-ß1 was released (mean ± SD in pg/mL of blood) from L-PRF (37,796 ± 5492) compared with L-PRP (23,738 ± 6848; p < 0.001) and blood clot (3739 ± 4690; p < 0.001), whereas more VEGF and IL-1ß were released from blood clot (1933 ± 704 and 2053 ± 908, respectively) compared with both L-PRP (642 ± 208; p < 0.001 and 273 ± 386; p < 0.001, respectively) and L-PRF (852 ± 376; p < 0.001 and 65 ± 56, p < 0.001, respectively). No differences were observed in IGF-1 and PDGF-AB released from any of the concentrates. TGF-ß1 release peaked at Day 7 in L-PRF and at 8 hours and Day 7 in L-PRP and 8 hours and Day 14 in blood clot. In all concentrates, main release of VEGF occurred between 3 and 7 days and of IL-1ß between Days 1 and 7. IGF-1 and PDGF-AB were released until Day 1 in L-PRP and blood clot, in contrast to sustained release over the first 3 days in L-PRF. The strongest migration of MSC occurred in response to L-PRF, and more HUVEC migration was seen in L-PRF and blood clot compared with L-PRP. TGF-ß1 correlated with initial platelet counts in L-PRF (Pearson r = 0.66, p = 0.0273) and initial leukocyte counts in L-PRP (Pearson r = 0.83, p = 0.0016). A positive correlation of IL-1ß on migration of MSC and HUVEC was revealed (Pearson r = 0.16, p = 0.0208; Pearson r = 0.31, p < 0.001). CONCLUSIONS: In comparison to L-PRP, L-PRF had higher amounts of released TGF-ß1, a long-term release of growth factors, and stronger induction of cell migration. Future preclinical studies should confirm these data in a defined injury model. CLINICAL RELEVANCE: By characterizing the biologic properties of different platelet concentrates in vitro, we may gain a better understanding of their clinical effects and develop guidelines for specific future applications.

S100A1 and S100B expression patterns identify differentiation status of human articular chondrocytes.

Many studies in the field of cell-based cartilage repair have focused on identifying markers associated with the differentiation status of human articular chondrocytes (HAC) that could predict their chondrogenic potency. A previous study from our group showed a correlation between the expression of S100 protein in HAC and their chondrogenic potential. The aims of the current study were to clarify which S100 proteins are associated with HAC differentiation status and to provide an S100-based assay for measuring HAC chondrogenic potential. The expression patterns of S100A1 and S100B were investigated in cartilage and in HAC cultured under conditions promoting dedifferentiation (monolayer culture) or redifferentiation (pellet culture or BMP4 treatment in monolayer culture), using characterized antibodies specifically recognizing S100A1 and S100B, by immunohistochemistry, immunocytochemistry, Western blot, and gene expression analysis. S100A1 and S100B were expressed homogeneously in all cartilage zones, and decreased during dedifferentiation. S100A1, but not S100B, was re-expressed in pellets and co-localized with collagen II. Gene expression analysis revealed concomitant modulation of S100A1, S100B, collagen type II, and aggrecan: down-regulation during monolayer culture and up-regulation upon BMP4 treatment. These results strongly support an association of S100A1, and to a lesser extent S100B, with the HAC differentiated phenotype. To facilitate their potential application, we established an S100A1/B-based flow cytometry assay for accurate assessment of HAC differentiation status. We propose S100A1 and S100B expression as a marker to develop potency assays for cartilage regeneration cell therapies, and as a redifferentiation readout in monolayer cultures aiming to investigate stimuli for chondrogenic induction.

A new histology scoring system for the assessment of the quality of human cartilage repair: ICRS II.

BACKGROUND: A reliable and reproducible method is needed to assess cartilage repair. PURPOSE: This study was undertaken to test the reproducibility of 2 established histological scoring systems, the Modified O'Driscoll Scale (MODS) and International Cartilage Research Society (ICRS) Visual Assessment Scale (ICRS I), and subsequently to develop and evaluate a new grading system for cartilage repair. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: A total of 107 cartilage biopsy specimens were graded using MODS and ICRS I, and the reader variability was measured. The new grading system, ICRS II, was developed and the inter- and intrareader variability determined by 3 independent readers. Collagen type II deposition was assessed immunohistochemically. RESULTS: The MODS and ICRS I demonstrated high interreader variability, with MODS also showing high intrareader variability. A new histological scoring system, ICRS II, was developed comprising 14 criteria to assess parameters related to chondrocyte phenotype and tissue structure. The ICRS II demonstrated lower inter- and intrareader variability compared with MODS or ICRS I. The overall assessment and matrix staining scores had the best correlation coefficients for inter- and intrareader variability (r = .81 and .82, respectively). The extent of collagen type II in cartilage, considered a marker of differentiation toward hyaline cartilage, could represent a measure of good cartilage repair. A correlation coefficient of .56 was obtained between the extent of collagen type II staining and the overall assessment score. CONCLUSION: The ICRS II represents an improvement over current histological cartilage repair grading systems in terms of reader reproducibility. The clinical relevance and its ability to predict long-term repair durability will be assessed once long-term clinical data become available.

Neocartilage formation in 1 g, simulated, and microgravity environments: implications for tissue engineering.

AIM: The aim of this study was to analyze and compare the deposition of cartilage-specific extracellular matrix components and cellular organization in scaffold-free neocartilage produced in microgravity and simulated microgravity. METHODS: Porcine chondrocytes were seeded (100 x 10(6)/mL) into cylindrical culture chambers (n = 8) and cultured in the following environments: (i) microgravity during the Flight 7S (Cervantes mission) on the International Space Station (ISS), (ii) simulated microgravity in a random positioning machine (RPM), and (iii) normal gravity (1 g, control). After 16 days, each neocartilage tissue was processed for histology, immunohistochemistry, quantitative real-time reverse transcriptase-polymerase chain reaction, and histomorphometric analysis. RESULTS: Weaker extracellular matrix staining of ISS neocartilage tissue was noted compared with both Earth-cultivated tissues. Higher collagen II/I expression ratios were observed in ISS samples compared with control tissue. Conversely, higher aggrecan/versican gene expression profiles were seen in control 1 g samples compared with microgravity samples. Cell density produced in microgravity was significantly reduced compared with the normal gravity neocartilage tissues. CONCLUSION: Tissue cultivated on the RPM showed intermediate characteristics compared with ISS and 1 g conditions. These data indicate that the RPM system does not sustain microgravity. Although microgravity impacts the development of in vitro generated cartilage, simulated microgravity using the RPM may be a useful tool to produce cartilaginous tissue grafts with fewer cells.

Population doublings and percentage of S100-positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes.

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57-4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90-negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90-positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100-positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell-based cartilage repair treatments.

The nuclear factor-kappaB and p53 pathways function independently in primary cells and transformed fibroblasts responding to genotoxic damage.

With nuclear factor-kappaB (NF-kappaB) and p53 functions generally having disparate outcomes for cell survival and cell division, understanding how these pathways are coordinated following a common activation signal such as DNA damage has important implications for cancer therapy. Conflicting reports concerning NF-kappaB and p53 interplay in different cell line models prompted a reexamination of this issue using mouse primary thymocytes and embryonic fibroblasts, plus fibroblasts transformed by E1A12S. Here, we report that following the treatment of these cells with a range of stress stimuli, p53 and NF-kappaB were found to regulate cell cycling and survival independently.

Immunophenotypic changes of human articular chondrocytes during monolayer culture reflect bona fide dedifferentiation rather than amplification of progenitor cells.

In this study, a time-course comparison of human articular chondrocytes (HAC) and bone marrow-derived mesenchymal stem cells (MSC) immunophenotype was performed in order to determine similarities/differences between both cell types during monolayer culture, and to identify HAC surface markers indicative of dedifferentiation. Our results show that dedifferentiated HAC can be distinguished from MSC by combining CD14, CD90, and CD105 expression, with dedifferentiated HAC being CD14+/CD90bright/CD105dim and MSC being CD14-/CD90dim/CD105bright. Surface markers on MSC showed little variation during the culture, whereas HAC showed upregulation of CD90, CD166, CD49c, CD44, CD10, CD26, CD49e, CD151, CD51/61, and CD81, and downregulation of CD49a, CD54, and CD14. Thus, dedifferentiated HAC appear as a bona fide cell population rather than a small population of MSC amplified during monolayer culture. While most of the HAC surface markers showed major changes at the beginning of the culture period (Passage 1-2), CD26 was upregulated and CD49a downregulated at later stages of the culture (Passage 3-4). To correlate changes in HAC surface markers with changes in extracellular matrix gene expression during monolayer culture, CD14 and CD90 mRNA levels were combined into a new differentiation index and compared with the established differentiation indices based on the ratios of mRNA levels of collagen type II to I (COL2/COL1) and of aggrecan to versican (AGG/VER). A correlation of CD14/CD90 ratio at the mRNA and protein level with the AGG/VER ratio during HAC dedifferentiation in monolayer culture validated CD14/CD90 as a new membrane and mRNA based HAC differentiation index.