Micro-fragmented adipose tissue cellular composition varies by processing device and analytical method.

Autologous adipose-derived biologics are of clinical interest based on accessibility of adipose tissue, a rich source of progenitor and immunomodulatory cells. Micro-fragmented adipose tissue (MFAT) preserves the cellular niche within intact extracellular matrix, potentially offering benefit over enzymatically-liberated stromal vascular fraction (SVF), however lack of standardized analyses complicate direct comparison of these products. In this study, MFAT from LipoGems® and AutoPose™ Restore systems, which utilize different washing and resizing methods, was analyzed for cellular content using different techniques. Flow cytometry was performed on SVF, with or without culture, and on the adherent cell population that naturally migrated from undigested MFAT. Cytokine release during culture was also assessed. SVF contained more diverse progenitor populations, while MFAT outgrowth contained lower cell concentrations of predominantly mesenchymal stromal cells (MSC). MSCs were significantly higher in MFAT from the AutoPose System for all analyses, with increased cytokine secretion characterized by high levels of anti-inflammatory and low to non-detectable inflammatory cytokines. These results demonstrate that cellularity depends on MFAT processing methods, and different techniques can be employed to evaluate graft cellularity. Comparisons of cell concentrations determined via these methods could be used to better interpret inter-study variability.

Autologous Fibrin Sealants Have Comparable Graft Fixation to an Allogeneic Sealant in a Biomechanical Cadaveric Model of Chondral Defect Repair.

Purpose: The purpose of this study is to assess the integrity of chondral defect repairs filled with a cartilage allograft and sealed with either allogeneic fibrin sealant or autologous fibrin sealants created with platelet-rich plasma (PRP) or platelet-poor plasma (PPP) in a cadaver model. Methods: Twenty-millimeter medial femoral condyle (MFC) chondral defects were created in five human cadaveric knees. The defects were filled with particulated cartilage allograft hydrated with PRP from human donors until slightly recessed. Sealants were applied until flush with the articular surface using PRP and autologous thrombin serum, PPP and autologous thrombin serum, or commercial allogeneic sealant. The MFC defects were cycled using a multiaxial testing system to simulate continuous passive motion undergone during rehabilitation. After testing, the repairs were assessed for integrity by quantitatively comparing defect exposure and qualitatively assessing sealant delamination. Results: The mean defect exposures were 4.20% ± 5.02% for the PRP group, 4.60% ± 5.18% for the PPP group, and 1.80% ± 2.95% for the allogeneic sealant group. No significant differences were observed between groups (P = .227), and each group had significantly less defect exposure when compared to the critical clinically relevant value assigned to be 30% (P = <.001 for all). No complete sealant delamination was observed, although the allogeneic sealant delaminated with a higher magnitude than did the autologous sealants. Conclusions: The PRP and PPP sealants were comparable to the allogeneic sealant for graft fixation when used in conjunction with an underlying PRP-hydrated particulated cartilage allograft. The autologous sealants had better delamination resistance than the allogeneic sealant. Clinical Relevance: The time-zero model is critical in elucidating the retention properties of fibrin and allogenic sealants after cartilage repair and before healing processes help stabilize the repair.

Bipolar Radiofrequency Ablation Does Not Result in Full-Thickness Articular Cartilage Penetration: An Ex Vivo Bovine Investigation.

Purpose: To evaluate the depth of penetration of manufacturer-recommended bipolar radiofrequency (BRF) output in healthy hyaline cartilage. Methods: Two matched knees from a bovine specimen were harvested for immediate testing. BRF probes were used to treat the articular cartilage in a hydrated noncontact technique employing a 1-mm spacer on patellar, condylar, and trochlear surfaces. Two manufacturer-recommended ablate power settings were evaluated to analyze the effect of varying power outputs on the depth of penetration. Surfaces were randomized and treated with BRF ablate setting 3 (AB-3), 4 (AB-4), or left untreated as a control (12 grids each). Slices were extracted from treatment zones and subjected to fluorescein diacetate and propidium iodide viability stains and analyzed with confocal light microscopy. A general linear model was used to determine whether variables such as ablation setting, cartilage location, and side significantly influenced depth of penetration (DoP) and cartilage thickness (Minitab 19, Chicago, IL). When significance was noted (P < .05), a post hoc-Tukey test was used to investigate specific differences. Results: AB-3 had a 50.9% lower mean DoP than AB-4 (P = .006). The mean DoP was 237.9 ± 140.6 µm for AB-3 and 484.1 ± 267.0 µm for AB-4. Median DoP values were 243.2 ± 149.5 µm for AB-3, 51.2% lower than the 498.4 ± 286.0 µm for AB-4. The mean maximum DoP for AB-3 was 302.4 ± 167.8 µm, 50.6% lower than AB-4 value of 611.6 ± 299.1 µm. Analysis of the cartilage thickness confirmed there was no difference in overall cartilage thickness used for AB-3 versus AB-4 testing (P = .953). Conclusions: The RF probe ablate power setting AB-3 demonstrated significantly less articular cartilage depth of penetration than the AB-4 setting in a healthy bovine model. Clinical Relevance: Debridement of chondral lesions with plasma BRF is of clinical interest. The presented study adds basic science information for those considering performing this technique.

Proteomic Analysis and Cell Viability of Nine Amnion, Chorion, Umbilical Cord, and Amniotic Fluid-Derived Products.

OBJECTIVE: Amnion products are used in various musculoskeletal surgeries and as injections for joint pain with conflicting reports of cell viability and protein contents. The objective of this study was to determine the full proteome and examine cell viability in 9 commercial amnion products using an unbiased bottom-up shotgun proteomics approach and confocal microscopy. DESIGN: Products were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and searched against a UniProt Homo sapiens database. Relative protein abundance was determined for each sample. Based on proteomics results, lumican was measured by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis was performed for interleukin-1 receptor antagonist (IL-1Ra) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). Cell viability was determined by calcein AM (live) and ethidium homodimer (dead) staining and confocal microscopy. RESULTS: Proteomic analysis revealed 919 proteins in the nine products. Proteins were primarily collagens, keratin, and albumin. Lumican, a small leucine-rich proteoglycan (SLRP) was found in all samples. Western blot analysis for IL-1Ra and TIMP-2 indicated presence of both proteins, with nonspecific antibody binding also present in all samples. No live cells were identified in any product. CONCLUSIONS: Several novel proteins were identified through proteomics that might impart the beneficial effects of amnion products, including SLRPs, collagens, and regulators of fibroblast activity. IL-1Ra and TIMP-2 were identified, but concentrations measured by ELISA may be falsely increased due to nonspecific antibody binding. The concept that the amnion tissues provide live cells to aid in tissue regeneration cannot be supported by the findings of this study.

Platelet-Rich Plasma Devices Can Be Used to Isolate Stem Cells From Synovial Fluid at the Point of Care.

PURPOSE: To assess whether point-of-care devices designed for collecting cellular components from blood or bone marrow could be used to isolate viable stem cells from synovial fluid. METHODS: Male and female patients older than 18 years old with either an acute, anterior cruciate ligament (ACL) injury or knee osteoarthritis (OA) with a minimum estimated 20 mL of knee effusion volunteered. Ten patients with an ACL injury and 10 patients with OA were enrolled. Two milliliters of collected synovial effusion were analyzed and cultured for cellular content. The remaining fluid was combined with whole blood and processed using a buffy-coat based platelet-rich plasma (PRP) processing system. Specimens were analyzed for cell counts, colony-forming unit (CFU) assays, differentiation assays, and flow cytometry. RESULTS: ACL effusion fluid contained 42.1 ± 20.7 CFU/mL and OA effusion fluid contained 65.4 ± 42.1 CFU/mL. After PRP processing, the counts in ACL-PRP were 101.6 ± 66.1 CFU/mL and 114.8 ± 73.4 CFU/mL in the OA-PRP. Cells showed tri-lineage differentiation potential when cultured under appropriate parameters. When analyzed with flow cytometry, >95% of cells produced with culturing expressed cell surface markers typically expressed by known stem cell populations, specifically CD45-, CD73+, CD29+, CD44+, CD105+, and CD90+. CONCLUSIONS: Multipotent viable stem cells can be harvested from knee synovial fluid, associated with an ACL injury or OA, and concentrated with a buffy coat-based PRP-processing device. CLINICAL RELEVANCE: PRP devices can be used to harvest stem cells from effusion fluids. Methods to use effusion fluid associated with an ACL injury and OA should be investigated further.

Autologous thrombin preparations: Biocompatibility and growth factor release.

Platelet-rich plasma (PRP) has been investigated to promote wound healing in a variety of tissues. Thrombin, another essential component of wound healing, is sometimes combined with PRP to generate a fibrin clot in order to retain the sample at the delivery site and to stimulate growth factor release. Using a fully autologous approach, autologous serum (AS) with thrombin activity can be prepared using a one-step procedure by supplementing with ethanol (E+ AS) to prolong room temperature stability or prepared ethanol free (E- AS) by utilizing a two-step procedure to prolong stability. The objective of this study was to evaluate potential wound healing mechanisms of these two preparations using commercially available devices. A variety of tests were conducted to assess biocompatibility and growth factor release from PRP at various ratios. It was found that E- AS contained greater leukocyte viability in the product (97.1 ± 2.0% compared to 41.8 ± 11.5%), supported greater bone marrow mesenchymal stem cell proliferation (3.7× vs 0.8× at a 1:4 ratio and 3.6× vs 1.6× at a 1:10 ratio), and stimulated release of growth factors and cytokines from PRP to a greater extent than E+ AS. Of the 36 growth factors and cytokines evaluated, release of 27 of them were significantly reduced by the presence of ethanol in at least one of the tested configurations. It is concluded that the high concentrations of ethanol needed to stabilize point of care autologous thrombin preparations could be detrimental to normal wound healing processes.

Biological Mechanisms for Cartilage Repair Using a BioCartilage Scaffold: Cellular Adhesion/Migration and Bioactive Proteins.

Objective. BioCartilage is a desiccated, particulated cartilage allograft used for repair of focal cartilage defects. It is mixed with a biologic such as bone marrow concentrate (BMC), pressed into a contained defect, and sealed with fibrin glue. The objective of this study was to assess if BioCartilage could serve as a bioactive scaffold by affecting cellular adhesion, cellular migration, or the release interleukin-1 receptor antagonist protein (IL-1RA), and to identify its full proteomic makeup. Design. Cartilage explants were used to model confined defects. BioCartilage was mixed with BMC, grafted into defects, and sealed with 1 of 5 fibrin glues. Constructs were cultured for 24 or 48 hours and then processed for live/dead microscopy. Chondrocyte and mesenchymal stem cell (MSC) adhesion on BioCartilage was assessed using scanning electron microscopy. Conditioned medium from cultures and the biologics used in the study were assayed for IL-1RA. The protein footprint of BioCartilage was determined using bottom-up proteomics. Results. BioCartilage supported chondrocyte and MSC attachment within 24 hours, and cell viability was retained in all constructs at 24 and 48 hours. Fibrin glue did not inhibit cell attachment. BMC had the highest concentration of IL-1RA. Proteomics yielded 254 proteins, including collagens, proteoglycans, and several bioactive proteins with known anabolic roles including cartilage oligomeric matrix protein. Conclusions. This study suggests that BioCartilage has the chemical composition and architecture to support cell adherence and migration and to provide bioactive proteins, which together should have biologics advantages in cartilage repair beyond its role as a scaffold.

Quantification and Qualification of Stem Cells From Blood After Mobilization With Filgrastim, and Concentration Using a Platelet-Rich Plasma System.

PURPOSE: To determine the cellular composition of a product created with peripheral blood harvested after systemic mobilization with filgrastim and processed with one point-of-care blood concentrating system, i.e., a platelet-rich plasma (PRP) system. The second purpose was to compare mobilized platelet-rich plasma (M-PRP) with a concentrated bone marrow aspirate (cBMA) and a PRP created from the same subjects with the same PRP system. METHODS: Ten healthy volunteer subjects were recruited for collection and analysis of 3 tissue sources: non-treated peripheral blood, bone marrow aspirate, and filgrastim-mobilized peripheral blood, involving 4 doses of weight-based filgrastim. One point-of-care blood and bone marrow concentrating system was used to create 3 products: PRP, cBMA, and M-PRP. Automated hematologic analysis was performed on all products to quantify total red blood cells, white blood cells (WBCs), monocyte, platelet, and hematopoietic progenitor cell (HPC) concentrations. Flow cytometry was used to determine hematopoietic and mesenchymal progenitor cell populations. Lastly, concentrates were cultured and fibroblast colony-forming units (CFU-F) and morphology of adherent cells were evaluated. RESULTS: M-PRP contained a greater concentration of WBC (mean difference = 53.2 k/µL; P < .0001), monocytes (mean difference = 8.3 k/µL; P = .002), and a trend toward a greater concentration of HPC (mean difference = 200.5 /µL; P = .060) when compared with PRP. M-PRP contained a greater concentration of monocytes (mean difference = 5.5 k/µL; P = .017) and a trend toward a greater concentration of platelets (mean difference = 348 k/µL; P = .051) and HPC (mean difference = 193.4 /µL; P = .068) when compared with cBMA. M-PRP had a similar concentration of platelets to PRP (mean difference = 110 k/µL; P = .051) and PRP had a greater concentration than cBMA (mean difference = 458 k/µL; P = .003). cBMA remained the only product capable of producing CFU-Fs (446 ± 247 /mL) as neither the M-PRP nor PRP produced CFU-Fs. M-PRP produced colonies consistent with WBC. CONCLUSIONS: M-PRP, produced with filgrastim mobilized blood and a proprietary PRP system, contained more total WBCs, monocytes, platelets, and HPCs than cBMA and more WBCs, monocytes, and HPCs than PRP. CLINICAL RELEVANCE: Filgrastim mobilized PRP may be an alternative to cBMA for use as a point-of-care product for orthopaedic treatments.

The clot thickens: Autologous and allogeneic fibrin sealants are mechanically equivalent in an ex vivo model of cartilage repair.

Fibrin sealants are commonly used in cartilage repair surgeries to adhere cells or grafts into a cartilage defect. Both autologous and commercial allogeneic fibrin sealants are used in cartilage repair surgeries, yet there are no studies characterizing and comparing the mechanical properties of fibrin sealants from all-autologous sources. The objectives of this study were to investigate (i) the effect of fibrinogen and thrombin sources on failure mechanics of sealants, and (ii) how sealants affect the adhesion of particulated cartilage graft material (BioCartilage) to surrounding cartilage under physiological loading. Allogeneic thrombin and fibrinogen were purchased (Tisseel), and autologous sources were prepared from platelet-rich plasma (PRP) and platelet-poor plasma (PPP) generated from human blood. To compare failure characteristics, sealants were sandwiched between cartilage explants and pulled to failure. The effect of sealant on the adhesion of BioCartilage graft to cartilage was determined by quantifying microscale strains at the graft-cartilage interface using an in vitro cartilage defect model subjected to shear loading at physiological strains well below failure thresholds. Fibrinogen sources were not equivalent; PRP fibrinogen created sealants that were more brittle, failed at lower strains, and resulted in sustained higher strains through the graft-cartilage interface depth compared to PPP and allogeneic sources. PPP clotted slower compared to PRP, suggesting PPP may percolate deeper into the repair to provide more stability through the tissue depth. There was no difference in bulk failure properties or microscale strains at the graft-cartilage interface between the purely autologous sealant (autologous thrombin + PPP fibrinogen) and the commercial allogeneic sealant. Clinical Significance: All-autologous fibrin sealants fabricated with PPP have comparable adhesion strength as commercial allogeneic sealants in vitro, whereas PRP creates an inferior all-autologous sealant that sustains higher strains through the graft-cartilage interface depth.

Exercise-Mobilized Platelet-Rich Plasma: Short-Term Exercise Increases Stem Cell and Platelet Concentrations in Platelet-Rich Plasma.

PURPOSE: To evaluate the effects of vigorous short-term exercise on the platelet and other cellular components of 2 point-of-care blood-processing devices: a buffy coat-based platelet-rich plasma (PRP) product and a plasma-based PRP product. METHODS: Twenty healthy subjects (aged 21-45 years) participated in a 20-minute vigorous exercise regimen on an upright stationary bike at 70% to 85% of maximum target heart rate. Pre- and post-exercise blood was processed in either a plasma-based or automated buffy coat-based PRP system. Complete blood counts were used to compare the cellular components in whole blood and the PRP products. RESULTS: Exercise significantly increased the concentrations of platelets by over 20% in whole blood (P < .001) and in both PRP products (P = .002 and P = .018). Both devices performed consistently with pre- and post-exercise blood. Buffy coat-based PRP prepared after exercise was also significantly larger in volume and had a significantly higher concentration of mobilized hematopoietic stem cells (hematopoietic progenitor cells [HPCs], from 1.7/µL to 2.7/µL, P = .043). The concentrations of all white blood cell types were increased, which could be differentially collected in the devices studied. CONCLUSIONS: Exercise can be used to consistently alter the composition of PRP. Twenty minutes of vigorous exercise can increase platelet concentrations in plasma-based and buffy coat-based PRP products and can increase HPC concentrations and volume in buffy coat-based PRP. CLINICAL RELEVANCE: This study shows a nonpharmacologic method to increase platelet and HPC harvests from peripheral blood. This is important because it highlights a method for altering biological therapies with limited comorbidity.

An Ethanol-Free Autologous Thrombin System.

Thrombin is a coagulation protein of central importance to hemostasis and wound healing that can be sourced from human blood, bovine blood, and engineered cell lines. Only autologous thrombin lacks the risks of transmitting emergent pathogens or eliciting an immunogenic response. Previous commercial autologous thrombin devices require the use of high concentrations of ethanol to achieve thrombin stability, introducing cytotoxicity risks. A new point of care device for preparing an ethanol-free autologous thrombin serum was investigated. The ethanol-free autologous serum (AS) was prepared using the Thrombinator™ System (Arthrex, Inc., Naples, FL). A total of 120 devices were tested with the blood of 30 healthy donors to determine the reliability and flexibility of the procedure. AS was prepared from both whole blood (WB) and platelet-poor plasma (PPP). Study endpoints were thrombin activity determined using a coagulation analyzer and formation of cohesive bone graft composites objectively measured using a durometer. The average thrombin activity produced by this system from 24 donors was 20.6 ± 2.7 IU/mL for WB and 13.4 ± 3.8 IU/mL for PPP which correlated to clot times of 3.9 and 5.9 seconds, respectively. The device tolerated use of varying volumes of blood to prepare AS. In addition, the system was able to generate four successive and comparable AS productions. When combined with platelet-rich plasma and bone graft material, cohesive scaffolds were always formed. A new device and method for preparing single donor, ethanol-free, AS with thrombin activity was demonstrated.

Approaches to improve integration and regeneration of an ex vivo derived temporomandibular joint disc scaffold with variable matrix composition.

Due to their natural biochemical and biomechanical characteristics, using ex vivo tissues as platforms for guided tissue regeneration has become widely accepted, however subsequent attachment and integration of these constructs in vivo is often overlooked. A decellularized porcine temporomandibular joint (TMJ) disc has shown promise as a scaffold to guide disc regeneration and preliminary work has shown the efficacy of surfactant (SDS) treatment within the fibrocartilaginous disc to remove cellular components. The majority of studies focus on the intermediate region of the disc (or disc proper). Using this approach, inherent attachment tissues can be maintained to improve construct stability and integration within the joint. Unlike human disc attachment tissue, the porcine attachment tissues have high lipid content which would require a different processing approach to remove immunogenic components. In order to examine the effect of delipidation on the attachment tissue properties, SDS and two organic solvent mixtures (acetone/ethanol and chloroform/methanol) were compared. Lipid and cellular solubilization, ECM alteration, and seeded human mesenchymal stem cell (MSC) morphology and viability were assessed. Quantitative analysis showed SDS treatments did not effectively delipidate the attachment tissues and cytotoxicity was noted toward MSC in these regions. Acetone/ethanol removed cellular material but not all lipids, while chloroform/methanol removed all visible lipid deposits but residual porcine cells were observed in histological sections. When a combination of approaches was used, no residual lipid or cytotoxicity was noted. Preparing a whole TMJ graft with a combined approach has the potential to improve disc integration within the native joint environment.

Mechanical Integrity of a Decellularized and Laser Drilled Medial Meniscus.

Since the meniscus has limited capacity to self-repair, creating a long-lasting meniscus replacement may help reduce the incidence of osteoarthritis (OA) after meniscus damage. As a first step toward this goal, this study evaluated the mechanical integrity of a decellularized, laser drilled (LD) meniscus as a potential scaffold for meniscal engineering. To evaluate the decellularization process, 24 porcine menisci were processed such that one half remained native tissue, while the other half was decellularized in sodium dodecyl sulphate (SDS). To evaluate the laser drilling process, 24 additional menisci were decellularized, with one half remaining intact while the other half was LD. Decellularization did not affect the tensile properties, but had significant effects on the cyclic compressive hysteresis and unconfined compressive stress relaxation. Laser drilling decreased the Young's modulus and instantaneous stress during unconfined stress relaxation and the circumferential ultimate strength during tensile testing. However, the losses in mechanical integrity in the LD menisci were generally smaller than the variance observed between samples, and thus, the material properties for the LD tissue remained within a physiological range. In the future, optimization of laser drilling patterns may improve these material properties. Moreover, reseeding the construct with cells may further improve the mechanical properties prior to implantation. As such, this work serves as a proof of concept for generating decellularized, LD menisci scaffolds for the purposes of meniscal engineering.

Biomechanical and biochemical outcomes of porcine temporomandibular joint disc deformation.

OBJECTIVE: The structure-function relationship in the healthy temporomandibular joint (TMJ) disc has been well established, however the changes in dysfunctional joints has yet to be systematically evaluated. Due to the poor understanding of the etiology of temporomandibular disorders (TMDs) this study evaluated naturally occurring degenerative remodeling in aged female porcine temporomandibular joint (TMJ) discs in order to gain insight into the progression and effects on possible treatment strategies of TMDs. DESIGN: Surface and regional biomechanical and biochemical properties of discal tissues were determined in grossly deformed (≥Wilkes Stage 3) and morphologically normal (≤Wilkes Stage 2) TMJ discs. RESULTS: Compared to normal disc structure the deformed discs lacked a smooth biconcave shape and characteristic ECM organization. Reduction in tensile biomechanical integrity and increased compressive stiffness and cellularity was found in deformed discs. Regionally, the posterior and intermediate zones of the disc were most frequently affected along with the inferior surface. CONCLUSIONS: The frequency of degeneration observed on the inferior surface of the disc (predominantly posterior), suggests that a disruption in the disc-condyle relationship likely contributes to the progression of joint dysfunction more than the temporodiscal relationship. As such, the inferior joint space may be an important consideration in early clinical diagnosis and treatment of TMDs, as it is overlooked in techniques performed in the upper joint space, including arthroscopy and arthrocentesis. Furthermore, permanent damage to the disc mechanical properties would limit the ability to successfully reposition deformed discs, highlighting the importance of emerging therapies such as tissue engineering.

The effect of terminal sterilization on structural and biophysical properties of a decellularized collagen-based scaffold; implications for stem cell adhesion.

Terminal sterilization induces physical and chemical changes in the extracellular matrix (ECM) of ex vivo-derived biomaterials due to their aggressive mechanism of action. Prior studies have focused on how sterilization affects the mechanical integrity of tissue-based biomaterials but have rarely characterized effects on early cellular interaction, which is indicative of the biological response. Using a model fibrocartilage disc scaffold, these investigations compare the effect of three common sterilization methods [peracetic acid (PAA), gamma irradiation (GI), and ethylene oxide (EtO)] on a range of material properties and characterized early cellular interactions. GI and EtO produced unfavorable structural damage that contributed to inferior cell adhesion. Conversely, exposure to PAA resulted in limited structural alterations while inducing chemical modifications that favored cell attachment. Results suggest that the sterilization approach can be selected to modulate biomaterial properties to favor cellular adhesion and has relevance in tissue engineering and regenerative medicine applications. Furthermore, the study of cellular interactions with modified biomaterials in vitro provides information of how materials may react in subsequent clinical applications.

Autologous protein solution prepared from the blood of osteoarthritic patients contains an enhanced profile of anti-inflammatory cytokines and anabolic growth factors.

The objective of this clinical study was to test if blood from osteoarthritis (OA) patients (n = 105) could be processed by a device system to form an autologous protein solution (APS) with preferentially increased concentrations of anti-inflammatory cytokines compared to inflammatory cytokines. To address this objective, APS was prepared from patients exhibiting radiographic evidence of knee OA. Patient metrics were collected including: demographic information, medical history, medication records, and Knee Injury and Osteoarthritis Outcome Score (KOOS) surveys. Cytokine and growth factor concentrations in whole blood and APS were measured using enzyme-linked immunosorbent assays. Statistical analyses were used to identify relationships between OA patient metrics and cytokines. The results of this study indicated that anti-inflammatory cytokines were preferentially increased compared to inflammatory cytokines in APS from 98% of OA patients. APS contained high concentrations of anti-inflammatory proteins including 39,000 ± 20,000 pg/ml IL-1ra, 21,000 ± 5,000 pg/ml sIL-1RII, 2,100 ± 570 pg/ml sTNF-RI, and 4,200 ± 1,500 pg/ml sTNF-RII. Analysis of the 82 patient metrics indicated that no single patient metric was strongly correlated (R(2) > 0.7) with the key cytokine concentrations in APS. Therefore, APS can be prepared from a broad range of OA patients.

Decellularization method influences early remodeling of an allogenic tissue scaffold.

Extracellular matrix-based biomaterials are currently pursued as an alternative to autologous transplants for the treatment of gingival recession and periodontal disease. These grafts offer improved tissue regeneration without the need for a second operative procedure used in current treatments to remove nonresorbable synthetic biomaterials. However, while decellularization is necessary to minimize the potential immunological impact, it can significantly modify the materials architectural and biochemical properties. By understanding cellular responses, it is possible to more specifically target varying clinical situations. These investigations assess a novel allogenic scaffold derived from the human umbilical vein and determine the effects of two decellularization approaches (osmotic lysis and the surfactant Triton X-100) on the biological and mechanical properties during early remodeling events. Results show Triton X-100 to be significantly more effective at extracting lipids, while the extraction of the scaffolds bulk protein, GAG and DNA similar between the two treatments. Once seeded, scaffolds prepared with osmotic lysis displayed increased cellular proliferation and reduced metabolic activity compared to scaffolds treated with surfactant. Biomechanical properties were largely preserved and similar between the two treatments. These results suggest that by optimizing scaffold processing conditions, biological events associated with remodeling can be modulated to tailor scaffold function for specific clinical applications.

Autologous solution protects bovine cartilage explants from IL-1α- and TNFα-induced cartilage degradation.

Osteoarthritis (OA) is characterized by deterioration of articular cartilage driven by an imbalance of pro- and anti-inflammatory cytokines. To address the cartilage deterioration observed in OA, an autologous protein solution (APS) has been developed which has been shown to inhibit the production of destructive proteases and inflammatory cytokines from chondrocytes and monocytes, respectively. The purpose of this study was to determine the chondroprotective effect of APS on IL-1α- or TNFα-challenged bovine articular cartilage explants. Cartilage explants were cultured in the presence or absence of recombinant inflammatory cytokines, IL-1α and TNFα. Explants under equivalent inflammatory conditions were pretreated with recombinant antagonists IL-1ra, sTNF-RI, or APS to measure their inhibition of matrix degradation. Explants were further evaluated with Safranin-O, Masson's Trichrome, and Hematoxylin and Eosin histological staining. APS was more effective than recombinant antagonists in preventing cartilage matrix degradation and inhibited any measurable IL-1α-induced collagen release over a 21-day culture period. APS treatment reduced the degree of Safranin-O staining loss when cartilage explants were cultured with IL-1α or TNFα. Micrographs of APS treated cartilage explants showed an increase in observed cellularity and apparent cell division. APS may have the potential to prevent cartilage loss associated with early OA.

Autologous protein solution inhibits MMP-13 production by IL-1ß and TNFα-stimulated human articular chondrocytes.

Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet-rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF-ß1, TGF-ß2, EGF, IGF-1, PDGF-AB, PDGF-BB, and VEGF) and anti-inflammatory (IL-1ra, sTNF-RI, sTNF-RII, IL-4, IL-10, IL-13, and IFNγ) cytokines but low concentrations of catabolic cytokines (IL-1α, IL-1ß, TNFα, IL-6, IL-8, IL-17, and IL-18). Human articular chondrocytes were pre-incubated with the antagonists IL-1ra, sTNF-RI, or APS prior to the addition of recombinant human IL-1ß or TNFα. Following exposure to inflammatory cytokines, the levels of MMP-13 in the culture medium were evaluated by ELISA. MMP-13 production stimulated in chondrocytes by IL-1ß or TNFα was reduced by rhIL-1ra and sTNF-RI to near basal levels. APS was also capable of inhibiting the production of MMP-13 induced by both IL-1ß and TNFα. The combination of anabolic and anti-inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA.