Randomized, Placebo-Controlled Analysis of the Knee Synovial Environment Following Platelet-Rich Plasma Treatment for Knee Osteoarthritis.

BACKGROUND: Platelet-rich-plasma (PRP) is used to treat knee osteoarthritis; however, mechanistic evidence of PRP effectiveness for pain relief is limited. OBJECTIVE: To assess molecular biomarkers and mesenchymal stem cells (MSCs) in synovial fluid during PRP treatment of the osteoarthritic knee joint. DESIGN: Single blinded, randomized, placebo controlled pilot study. SETTING: Veterans Affairs Medical Center. PARTICIPANTS: Seventeen participants with mild to moderate knee osteoarthritis were randomized in a 2:1 placebo-controlled ratio, receiving PRP or saline (placebo) intra-articular injection into the knee joint. METHODS: Knee synovial fluid was analyzed before the respective injections and again 10 days following injection. Participants were followed up to 12 months completing visual analog scale (VAS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaires at intervals over that period. MAIN OUTCOME MEASURES: The effects of PRP on synovial protein and MSC gene expression levels were measured by multiplex enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. RESULTS: Novel biomarkers including levels of interleukin (IL)-5, IL-6, IL-10, and tumor necrosis factor-α were measured in synovial fluid 10 days after PRP treatment. Altered gene expression profiles in MSCs from patients treated with PRP were observed for matrix metalloproteinases and inflammatory markers (IL-6, IL-8, CCL2, TNF-α). A2M protease was significantly increased following PRP treatment (P = .005). WOMAC scores declined for up to 3 months from baseline levels and remained low at 6 and 12 months in the PRP group. In contrast, WOMAC scores for patients receiving the saline injection were relatively unchanged for up to 12 months. CONCLUSIONS: We report significant changes for the biomarker A2M (P = .005) as well as differences in expression of cellular markers and postulate that PRP modulates the local knee synovial environment by altering the inflammatory milieu, matrix degradation, and angiogenic growth factors. The PRP treatment group had less pain and stiffness and improved function scores.

Mechanical Strain of the Trilobed Transposition Flap in Artificial Skin Models: Pivotal Restraint Decreases With Decreasing Rotational Angles.

BACKGROUND: In transposition flaps, thicker tissue and higher degrees of rotation are associated with increased pivotal restraint; however, limited experimental data exist quantifying the degree to which these affect flap biomechanics. The use of artificial skin models in conjunction with digital image correlation technology allows for investigation into biomechanical properties of skin flaps. OBJECTIVE: To quantify the effects of tissue thickness and rotational angles on pivotal restraint within transposition flaps using artificial skin models. METHODS: Ninety degree bilobed and trilobed flaps were used to close defects in artificial skin models of increasing thicknesses. Digital image correlation was used to quantify strain. Quantitative and qualitative differences in strain were assessed in increasing flap thicknesses and between flap designs. RESULTS: Increasing flap thickness was associated with decreasing strain. In the bilobed flap, increasing thickness was associated with displacement of the flap pivot point away from the distal flap edge. Comparatively, lower angles of rotation in the trilobed flap were not associated with migration of the flap pivot point. CONCLUSION: Increased pivotal restraint observed in higher degrees of rotation is due to migration of the flap pivot point. This model supports the common practice of decreasing flap angles to compensate for pivotal restraint.

Lactic acid suppresses IgE-mediated mast cell function in vitro and in vivo.

Mast cells have functional plasticity affected by their tissue microenvironment, which greatly impacts their inflammatory responses. Because lactic acid (LA) is abundant in inflamed tissues and tumors, we investigated how it affects mast cell function. Using IgE-mediated activation as a model system, we found that LA suppressed inflammatory cytokine production and degranulation in mouse peritoneal mast cells, data that were confirmed with human skin mast cells. In mouse peritoneal mast cells, LA-mediated cytokine suppression was dependent on pH- and monocarboxylic transporter-1 expression. Additionally, LA reduced IgE-induced Syk, Btk, and ERK phosphorylation, key signals eliciting inflammation. In vivo, LA injection reduced IgE-mediated hypothermia in mice undergoing passive systemic anaphylaxis. Our data suggest that LA may serve as a feedback inhibitor that limits mast cell-mediated inflammation.

Preliminary investigation of honey-doped electrospun scaffolds to delay wound closure.

Manuka honey is an ancient remedy to improve wound healing; however, an effective delivery system is needed to facilitate extended release of honey into wounds. We developed an electrospun dermal regeneration template consisting of a poly (ε-caprolactone) (PCL) scaffold embedded with 1%, 5%, 10%, or 20% manuka honey. In vitro studies demonstrated that honey PCL scaffolds were not toxic to macrophages, and they allowed for macrophage infiltration into the scaffolds. Vascular endothelial growth factor (VEGF), a marker of angiogenesis, was released by macrophages cultured with scaffolds and macrophage/scaffold conditioned media promoted endothelial cell tube formation in an angiogenesis assay. In a full thickness murine wound model, the scaffolds prevented rapid wound contraction. In vivo, cells infiltrated the scaffolds by post-wounding day 7, but the honey scaffolds did not affect collagen deposition at that time. In summary, preliminary studies investigating the effect of honey on tissue repair show that scaffolds prevent rapid wound contraction, allow for cell infiltration, and promote angiogenesis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2620-2628, 2019.

Insert-based microfluidics for 3D cell culture with analysis.

We present an insert-based approach to fabricate scalable and multiplexable microfluidic devices for 3D cell culture and integration with downstream detection modules. Laser-cut inserts with a layer of electrospun fibers are used as a scaffold for 3D cell culture, with the inserts being easily assembled in a 3D-printed fluidic device for flow-based studies. With this approach, the number and types of cells (on the inserts) in one fluidic device can be customized. Moreover, after an investigation (i.e., stimulation) under flowing conditions, the cell-laden inserts can be removed easily for subsequent studies including imaging and cell lysis. In this paper, we first discuss the fabrication of the device and characterization of the fibrous inserts. Two device designs containing two (channel width = 260 µm) and four (channel width = 180 µm) inserts, respectively, were used for different experiments in this study. Cell adhesion on the inserts with flowing media through the device was tested by culturing endothelial cells. Macrophages were cultured and stimulated under different conditions, the results of which indicate that the fibrous scaffolds under flow conditions result in dramatic effects on the amount and kinetics of TNF-α production (after LPS stimulation). Finally, we show that the cell module can be integrated with a downstream absorbance detection scheme. Overall, this technology represents a new and versatile way to culture cells in a more in vivo fashion for in vitro studies with online detection modules. Graphical abstract This paper describes an insert-based microfluidic device for 3D cell culture that can be easily scaled, multiplexed, and integrated with downstream analytical modules.

A preliminary in vitro evaluation of the bioactive potential of cryogel scaffolds incorporated with Manuka honey for the treatment of chronic bone infections.

Previous studies have identified honey as an agent in bacterial inhibition and a mediator in lowering the pH at the wound site. Manuka honey (MH), indigenous to New Zealand, contains a Unique Manuka Factor that provides an additional antibacterial agent. While there are many potential benefits to incorporating MH into wounds, there is currently no ideal way to deliver the material to the site of injury. Cryogels are a type of scaffold that possess high porosity, mechanical stability, and a sponge-like consistency. This study uniquely incorporates varying amounts of MH into cryogel scaffolds, utilizing its properties in a sustained release fashion to assist in the overall healing process, while using the cryogel structure as a tissue template. All cryogels were evaluated to determine the effects of MH on porosity, swelling potential, mechanical durability, and cell compatibility. The release of MH was also quantified to evaluate bacterial clearance potential, and the scaffolds were mineralized to replicate native bone. It was determined that a 5% MH silk fibroin cryogel has the potential to inhibit bacterial growth while still maintaining adequate porosity, mechanical properties, and cell infiltration. Such a scaffold would have use in a number of applications, including bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1918-1933, 2018.

Cryogel scaffolds from patient-specific 3D-printed molds for personalized tissue-engineered bone regeneration in pediatric cleft-craniofacial defects.

Bone defects are extremely common in children with cleft-craniofacial conditions, especially those with alveolar cleft defects and cranial defects. This study used patient-specific 3D-printed molds derived from computed tomography and cryogel scaffold fabrication as a proof of concept for the creation of site-specific implants for bone reconstruction. Cryogel scaffolds are unique tissue-engineered constructs formed at sub-zero temperatures. When thawed, the resulting structure is macroporous, sponge-like, and mechanically durable. Due to these unique properties, cryogels have excellent potential for the treatment of patient-specific bone defects; however, there is little literature on their use in cleft-craniofacial defects. While 3D-printing technology currently lacks the spatial resolution to print the microstructure necessary for bone regeneration, it can be used to create site-specific molds. Thus, it is ideal to integrate these techniques for the fabrication of scaffolds with patient-specific geometry. Overall, all cryogels possessed appropriate geometry to allow for cell infiltration after 28 days. Additionally, suitable mechanical durability was demonstrated where, despite mold geometry, all cryogels could be compressed without exhibiting crack propagation. Such a patient-specific scaffold would be ideal in pediatric cleft-craniofacial defects, as these are complex 3D defects and children have less donor bone availability.

The fabrication of cryogel scaffolds incorporated with poloxamer 407 for potential use in the regeneration of the nucleus pulposus.

Degeneration of the nucleus pulposus (NP) is the primary cause of back pain in almost 80% of the world population. The current gold standard treatment for a degenerated NP is a spinal fusion surgery which is costly, temporary, and extremely invasive. Research has been moving towards minimally invasive methods to lessen the collateral damage created during surgery. The use of a tissue-engineered scaffold has the potential to promote a healthy and hydrated environment to regenerate the NP. Cryogels are unique polymeric scaffolds composed of a highly connected, macroporous structure, and are capable of maintaining stability under high deformations. For this study, cryogels have been developed using gelatin and poloxamer 407 (P407) at varying ratios to determine the ideal combination of stability, water retention, and pore size. For the application of NP regeneration, a gelatin-P407 cryogel should be both stable and a well hydrated carrier. The cryogels created varied from a 1:1 gelatin to P407 ratio to a 10:1 ratio. The inclusion of P407 in the cryogels resulted in a significant increase in hydrophilicity, ideal pore size for cell infiltration, mechanical stability over 28 days, and cell infiltration after just 21 days. This novel gelatin-P407 composite cryogel has the potential to be a practical alternative to the spinal fusion procedure, saving patients hundreds of thousands of dollars and, ideally, leading to improved patient outcome.

Lactic Acid Suppresses IL-33-Mediated Mast Cell Inflammatory Responses via Hypoxia-Inducible Factor-1α-Dependent miR-155 Suppression.

Lactic acid (LA) is present in tumors, asthma, and wound healing, environments with elevated IL-33 and mast cell infiltration. Although IL-33 is a potent mast cell activator, how LA affects IL-33-mediated mast cell function is unknown. To investigate this, mouse bone marrow-derived mast cells were cultured with or without LA and activated with IL-33. LA reduced IL-33-mediated cytokine and chemokine production. Using inhibitors for monocarboxylate transporters (MCT) or replacing LA with sodium lactate revealed that LA effects are MCT-1- and pH-dependent. LA selectively altered IL-33 signaling, suppressing TGF-ß-activated kinase-1, JNK, ERK, and NF-κB phosphorylation, but not p38 phosphorylation. LA effects in other contexts have been linked to hypoxia-inducible factor (HIF)-1α, which was enhanced in bone marrow-derived mast cells treated with LA. Because HIF-1α has been shown to regulate the microRNA miR-155 in other systems, LA effects on miR-155-5p and miR-155-3p species were measured. In fact, LA selectively suppressed miR-155-5p in an HIF-1α-dependent manner. Moreover, overexpressing miR-155-5p, but not miR-155-3p, abolished LA effects on IL-33-induced cytokine production. These in vitro effects of reducing cytokines were consistent in vivo, because LA injected i.p. into C57BL/6 mice suppressed IL-33-induced plasma cytokine levels. Lastly, IL-33 effects on primary human mast cells were suppressed by LA in an MCT-dependent manner. Our data demonstrate that LA, present in inflammatory and malignant microenvironments, can alter mast cell behavior to suppress inflammation.

In vitro characterization of MG-63 osteoblast-like cells cultured on organic-inorganic lyophilized gelatin sponges for early bone healing.

The development of three-dimensional porous scaffolds with enhanced osteogenic and angiogenic potential would be beneficial for inducing early-stage bone regeneration. Previous studies have demonstrated the advantages of mineralized and nonmineralized acellular 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) cross-linked gelatin sponges enhanced with preparations rich in growth factors, hydroxyapatite, and chitin whiskers. In this study, those same scaffolds were mineralized and dynamically seeded with MG-63 cells. Cell proliferation, protein/cytokine secretion, and compressive mechanical properties of scaffolds were evaluated. It was found that mineralization and the addition of growth factors increased cell proliferation compared to gelatin controls. Cells on all scaffolds responded in an appropriate bone regenerative fashion as shown through osteocalcin secretion and little to no secretion of bone resorbing markers. However, compressive mechanical properties of cellularized scaffolds were not significantly different from acellular scaffolds. The combined results of increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion on scaffolds support the need for the addition of a bone-like mineral surface. Cellularized scaffolds containing growth factors reported similar advantages and mechanical values in the range of native tissues present in the early stages of bone healing. These results suggest that the developed composite sponges exhibited cellular responses and mechanical properties appropriate for promoting early bone healing in various applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2011-2019, 2016.

The influence of platelet-rich plasma on myogenic differentiation.

The ability to expand and direct both precursor and stem cells towards a differential fate is considered extremely advantageous in tissue engineering. Platelet-rich plasma (PRP) possesses a milieu of growth factors and cytokines, which have the potential to have either a differentiative or proliferative influence on the cell type tested. Here, we investigated the effect of PRP on C2C12 myoblasts. A range of PRP concentrations in differentiation media was used to determine whether a concentration dependence existed, while PRP embedded in fibres of aligned electrospun polydioxanone and polycaprolactone was used to determine whether this presence of fibres would cause any differences in response. In both cases, it was found that late myogenic markers were suppressed after 7 days in culture. However, an early differentiation marker, MyoD, was upregulated during this same time period. The results from this study represent the ability of PRP to have an influence over both myogenic proliferation and differentiation, a factor which could prove useful in future studies involved with skeletal muscle tissue engineering.

Mineralization Potential of Electrospun PDO-Hydroxyapatite-Fibrinogen Blended Scaffolds.

The current bone autograft procedure for cleft palate repair presents several disadvantages such as limited availability, additional invasive surgery, and donor site morbidity. The present preliminary study evaluates the mineralization potential of electrospun polydioxanone:nano-hydroxyapatite : fibrinogen (PDO : nHA : Fg) blended scaffolds in different simulated body fluids (SBF). Scaffolds were fabricated by blending PDO : nHA : Fg in the following percent by weight ratios: 100 : 0 : 0, 50 : 25 : 25, 50 : 50 : 0, 50 : 0 : 50, 0 : 0 : 100, and 0 : 50 : 50. Samples were immersed in (conventional (c), revised (r), ionic (i), and modified (m)) SBF for 5 and 14 days to induce mineralization. Scaffolds were characterized before and after mineralization via scanning electron microscopy, Alizarin Red-based assay, and modified burnout test. The addition of Fg resulted in scaffolds with smaller fiber diameters. Fg containing scaffolds also induced sheet-like mineralization while individual fiber mineralization was noticed in its absence. Mineralized electrospun Fg scaffolds without PDO were not mechanically stable after 5 days in SBF, but had superior mineralization capabilities which produced a thick bone-like mineral (BLM) layer throughout the scaffolds. 50 : 50 : 0 scaffolds incubated in either r-SBF for 5 days or c-SBF for 14 days produced scaffolds with high mineral content and individual-mineralized fibers. These mineralized scaffolds were still porous and will be further optimized as an effective bone substitute in future studies.

Electrospinning adipose tissue-derived extracellular matrix for adipose stem cell culture.

Basement membrane-rich extracellular matrices, particularly murine sarcoma-derived Matrigel, play important roles in regenerative medicine research, exhibiting marked cellular responses in vitro and in vivo, although with limited clinical applications. We find that a human-derived matrix from lipoaspirate fat, a tissue rich in basement membrane components, can be fabricated by electrospinning and used to support cell culture. We describe practical applications and purification of extracellular matrix (ECM) from adipose tissue (At-ECM) and its use in electrospinning scaffolds and adipose stem cell (ASC) culture. The matrix composition of this purified and electrospun At-ECM was assessed histochemically for basement membrane, connective tissue, collagen, elastic fibers/elastin, glycoprotein, and proteoglycans. Each histochemical stain was positive in fat tissue, purified At-ECM, and electrospun At-ECM, and to some extent positive in a 10:90 blend with polydioxanone (PDO). We also show that electrospun At-ECM, alone and blended with PDO, supports ASC attachment and growth, suggesting that electrospun At-ECM scaffolds support ASC cultivation. These studies show that At-ECM can be isolated and electrospun as a basement membrane-rich tissue engineering matrix capable of supporting stem cells, providing the groundwork for an array of future regenerative medicine advances.