Hyeon Joo Kim

Comparison of Growth Factor and Platelet Concentration From Commercial Platelet-Rich Plasma Separation Systems

Background: Clinical studies claim that platelet-rich plasma (PRP) shortens recovery times because of its high concentration of growth factors that may enhance the tissue repair process. Most of these studies obtained PRP using different separation systems, and few analyzed the content of the PRP used as treatment. Purpose: This study characterized the composition of single-donor PRP produced by 3 commercially available PRP separation systems. Study Design: Controlled laboratory study. Methods: Five healthy humans donated 100 mL of blood, which was processed to produce PRP using 3 PRP concentration systems (MTF Cascade, Arteriocyte Magellan, Biomet GPS III). Platelet, white blood cell (WBC), red blood cell, and fibrinogen concentrations were analyzed by automated systems in a clinical laboratory, whereas ELISA determined the concentrations of platelet-derived growth factor αβ and ββ (PDGF-αβ, PDGF-ββ), transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor (VEGF). Results: There was no significant difference in mean PRP platelet, red blood cell, active TGF-β1, or fibrinogen concentrations among PRP separation systems. There was a significant difference in platelet capture efficiency. The highest platelet capture efficiency was obtained with Cascade, which was comparable with Magellan but significantly higher than GPS III. There was a significant difference among all systems in the concentrations of WBC, PDGF-αβ, PDGF-ββ, and VEGF. The Cascade system concentrated leukocyte-poor PRP, compared with leukocyte-rich PRP from the GPS III and Magellan systems. Conclusion: The GPS III and Magellan concentrate leukocyte-rich PRP, which results in increased concentrations of WBCs, PDGF-αβ, PDGF-ββ, and VEGF as compared with the leukocyte-poor PRP from Cascade. Overall, there was no significant difference among systems in the platelet concentration, red blood cell, active TGF-β1, or fibrinogen levels. Clinical Relevance: Products from commercially available PRP separation systems produce differing concentrations of growth factors and WBCs. Further research is necessary to determine the clinical relevance of these findings.

Bone Tissue Engineering with Premineralized Silk Scaffolds

Silk fibroin biomaterials are being explored as novel protein-based systems for cell and tissue culture. In the present study, biomimetic growth of calcium phosphate on porous silk fibroin polymeric scaffolds was explored to generate organic/inorganic composites as scaffolds for bone tissue engineering. Aqueous-derived silk fibroin scaffolds were prepared with the addition of polyaspartic acid during processing, followed by the controlled deposition of calcium phosphate by exposure to CaCl(2) and Na(2)HPO(4). These mineralized protein-composite scaffolds were subsequently seeded with human bone marrow stem cells (hMSC) and cultured in vitro for 6 weeks under osteogenic conditions with or without BMP-2. The extent of osteoconductivity was assessed by cell numbers, alkaline phosphatase and calcium deposition, along with immunohistochemistry for bone-related outcomes. The results suggest increased osteoconductive outcomes with an increase in initial content of apatite and BMP-2 in the silk fibroin porous scaffolds. The premineralization of these highly porous silk fibroin protein scaffolds provided enhanced outcomes for the bone tissue engineering.

The Effect of Platelet-Rich Plasma Formulations and Blood Products on Human Synoviocytes: Implications for Intra-articular Injury and Therapy

Background: The effect of platelet-rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture, but considerably less attention has been given to the effect of PRP on synoviocytes. Fibroblast-like synoviocytes (FLS) compose 80% of the normal human synovium and produce cytokines and matrix metalloproteinases that can mediate cartilage catabolism. Purpose: To compare the effects of leukocyte-rich PRP (LR-PRP), leukocyte-poor PRP (LP-PRP), red blood cell (RBC) concentrate, and platelet-poor plasma (PPP) on human FLS to determine whether leukocyte and erythrocyte concentrations of PRP formulations differentially affect the production of inflammatory mediators. Study Design: Controlled laboratory study. Methods: Peripheral blood was obtained from 4 donors and processed to create LR-PRP, LP-PRP, RBCs, and PPP. Human synoviocytes were cultured for 96 hours with the respective experimental conditions using standard laboratory conditions. Cell viability and inflammatory mediator production were then evaluated. Results: Treatment with LR-PRP resulted in significantly greater synoviocyte death (4.9% ± 3.1%) compared with LP-PRP (0.72% ± 0.70%; P = .035), phosphate-buffered saline (PBS) (0.39% ± 0.27%; P = .018), and PPP (0.26% ± 0.30%; P = .013). Synoviocytes treated with RBC concentrate demonstrated significantly greater cell death (12.5% ± 6.9%) compared with PBS (P < .001), PPP (P < .001), LP-PRP (P < .001), and LR-PRP (4.9% ± 3.1%; P < .001). Interleukin (IL)–1β content was significantly higher in cultures treated with LR-PRP (1.53 ± 0.86 pg/mL) compared with those treated with PBS (0.22 ± 0.295 pg/mL; P < .001), PPP (0.11 ± 0.179 pg/mL; P < .001), and RBCs (0.64 ± 0.58 pg/mL; P = .001). IL-6 content was also higher with LR-PRP (32,097.82 ± 22,844.300 pg/mL) treatment in all other groups (P < .001). Tumor necrosis factor–α levels were greatest in LP-PRP (9.97 ± 3.110 pg/mL), and this was significantly greater compared with all other culture conditions (P < .001). Interferon-γ levels were greatest in RBCs (64.34 ± 22.987 pg/mL) and significantly greater than all other culture conditions (P < .001). Conclusion: Treatment of synovial cells with LR-PRP and RBCs resulted in significant cell death and proinflammatory mediator production. Clinical Relevance: Clinicians should consider using leukocyte-poor, RBC-free formulations of PRP when administering intra-articularly.

Relationships between degradability of silk scaffolds and osteogenesis

Bone repairs represent a major focus in orthopedic medicine with biomaterials as a critical aspect of the regenerative process. However, only a limited set of biomaterials are utilized today and few studies relate biomaterial scaffold design to degradation rate and new bone formation. Matching biomaterial remodeling rate towards new bone formation is important in terms of the overall rate and quality of bone regeneration outcomes. We report on the osteogenesis and metabolism of human bone marrow derived mesenchymal stem cells (hMSCs) in 3D silk scaffolds. The scaffolds were prepared with two different degradation rates in order to study relationships between matrix degradation, cell metabolism and bone tissue formation in vitro. SEM, histology, chemical assays, real-time PCR and metabolic analyses were assessed to investigate these relationships. More extensively mineralized ECM formed in the scaffolds designed to degrade more rapidly, based on SEM, von Kossa and type I collagen staining and calcium content. Measures of osteogenic ECM were significantly higher in the more rapidly degrading scaffolds than in the more slowly degrading scaffolds over 56 days of study in vitro. Metabolic analysis, including glucose and lactate levels, confirmed the degradation rate differences with the two types of scaffolds, with the more rapidly degrading scaffolds supporting higher levels of glucose consumption and lactate synthesis by the hMSCs upon osteogenesis, in comparison to the more slowly degrading scaffolds. The results demonstrate that scaffold degradation rates directly impact the metabolism of hMSCs, and in turn the rate of osteogenesis. An understanding of the interplay between cellular metabolism and scaffold degradability should aid in the more rational design of scaffolds for bone regeneration needs both in vitro and in vivo.

The In Vitro Chondrotoxicity of Single-Dose Local Anesthetics

Background: The administration of amide-type local anesthetics to cartilaginous tissues has revealed potential chondrotoxicity. Purpose: This study evaluated whether administration of single doses of 1% lidocaine, 0.25% bupivacaine, and 0.5% ropivacaine resulted in decreased chondrocyte viability or cartilage matrix degradation in vitro. Study Design: Controlled laboratory study. Methods: Monolayer human chondrocytes and intact cartilage samples were cultured for 1 week in media. Each drug was delivered in a custom bioreactor over its clinical duration of action. A Live/Dead Viability/Cytotoxicity Assay was used to determine the ratio of dead to live cells for monolayer chondrocyte cultures compared with controls. Damage to the cartilage extracellular matrix (ECM) in en bloc cartilage samples was evaluated by analysis of DNA, glycosaminoglycan (GAG), and collagen content. Results: Chondrocytes treated for 3 hours with a single dose of 1% lidocaine exhibited significantly more cell death (7.9%) compared with control media (2.9%; P < .001). No significant difference in cell death was observed in chondrocytes treated for 6 hours with 0.25% bupivacaine (2.7%) versus controls (2.8%; P = .856) or cells treated for 12 hours in 0.5% ropivacaine (2.9%) versus controls (2.4%; P = .084). There was no significant difference in GAG expression (P = .627) or DNA-normalized GAG expression (P = .065) between the intact cartilage treatment groups; however, the DNA-normalized GAG expression was markedly lower in cartilage cultures treated with 1% lidocaine (3.36 ± 1.15) compared with those in control media (7.61 ± 3.83). Conclusion: The results of this in vitro study indicate that a single-dose administration of 1% lidocaine resulted in a significant decrease in chondrocyte viability when compared with control cultures. Clinical Relevance: Single-dose injections of 1% lidocaine may be significantly chondrotoxic, and further investigation regarding in vivo chondrotoxicity appears warranted.

Enhancing annulus fibrosus tissue formation in porous silk scaffolds.

There is presently no optimal treatment for patients with chronic back pain as a result of degenerative disc disease. Tissue engineering, an annulus fibrosus (AF) construct suitable to repair the damaged AF, is one novel approach to the treatment of this disease. We have previously demonstrated that porous silk scaffolds can support AF cell attachment and extracellular matrix accumulation; however, tissue infiltration and matrix accumulation was not optimal. The purpose of this study was to determine whether the dynamic culture of AF cells seeded into larger average pore size silk scaffolds would improve tissue formation. AF cells were isolated from bovine caudal discs and seeded into porous silk scaffolds and grown in either dynamic or static flow conditions. The cell-seeded scaffolds were grown for up to 4 weeks and evaluated for cell attachment, gene expression, histological appearance, and matrix accumulation. Dynamic culture improved AF tissue formation as the tissue was more cellular and contained significantly more matrix than that formed in static culture. Spatial distribution of tissue was comparable for static and dynamic culture. Varying scaffold pore sizes (200-, 600-, and 1000-microm pore size) demonstrated that an average pore size of 600 microm resulted in the most uniform tissue distribution with the greatest amount of type I collagen. Our study suggests that dynamic flow conditions and scaffold pore size can affect the formation of engineered AF tissue.

Non-invasive characterization of structure and morphology of silk fibroin biomaterials using non-linear microscopy.

Designing biomaterial scaffolds remains a major challenge in tissue engineering. Key to this challenge is improved understanding of the relationships between the scaffold properties and its degradation kinetics, as well as the cell interactions and the promotion of new matrix deposition. Here we present the use of non-linear spectroscopic imaging as a non-invasive method to characterize not only morphological, but also structural aspects of silkworm silk fibroin-based biomaterials, relying entirely on endogenous optical contrast. We demonstrate that two photon excited fluorescence and second harmonic generation are sensitive to the hydration, overall beta sheet content and molecular orientation of the sample. Thus, the functional content and high resolution afforded by these non-invasive approaches offer promise for identifying important connections between biomaterial design and functional engineered tissue development. The strategies described also have broader implications for understanding and tracking the remodeling of degradable biomaterials under dynamic conditions both in vitro and in vivo.

Chondrotoxicity of Low pH, Epinephrine, and Preservatives Found in Local Anesthetics Containing Epinephrine

Background: Recent clinical and basic science investigations have revealed the chondrotoxicity of local anesthetics, especially those containing epinephrine, administered via an intra-articular pain pump. However, the exact mechanism of toxicity is unknown. This study evaluates the chondrotoxicity of low pH, epinephrine, and preservatives found in commonly used local anesthetics. Hypothesis: The chondrotoxicity of local anesthetics containing epinephrine is due to low pH, epinephrine, or the preservative sodium metabisulfite. Study Design: Controlled laboratory study. Methods: Human chondrocytes were harvested and cultured in a custom bioreactor designed to simulate metabolism of medication. Pain pumps were used to infuse one of the following medications into the culture system: control media; media titrated to pH 4.5, 5.0, 5.5, 6.0, 6.5; media with 1:100000 or 1:200000 epinephrine only; media with 0.5 mg/mL of sodium metabisulfite preservative; media with 0.5 mg/mL of methylparaben preservative, 0.25% bupivacaine, 0.25% bupivacaine with epinephrine, 1% lidocaine, and 1% lidocaine with epinephrine. Cultures were perfused for 24 hours and then were stained with live/dead cell viability assay. The chondrocytes were then examined by fluorescence microscopy and counted, and the percentage of cell death was calculated. Results: Cultures containing media titrated to pH 4.5 and 5.0 and local anesthetics containing epinephrine (pH 4.0-5.5) had high cell death rates compared with controls at all time points (P < .001), while cultures containing 1:100000 and 1:200000 epinephrine alone had no increased death rate. Also, 0.5 mg/mL sodium metabisulfite preservative had a significant effect on cell death (P < .034); however, the preservative methylparaben had no effect (P > .05). The percentage of cell death was not significant for 1% lidocaine (12.5%; P > .943) and 0.25% bupivacaine (16.5%; P > .609). Conclusion: The marked chondrotoxicity of local anesthetics containing epinephrine appears to be a combined effect of low pH, as these medications are titrated to pH 4.0 to 5.5 for product stability, and the preservative sodium metabisulfite. Extreme caution should be exercised when using intra-articular pain pumps with local anesthetics containing epinephrine. Clinical Relevance: Understanding the causes of chondrotoxicity using local anesthetics containing epinephrine is critical to decrease complications associated with this class of medications.

Processing Windows for Forming Silk Fibroin Biomaterials into a 3D Porous Matrix

In the present study we clarify phase diagrams related to silk fibroin processing into three-dimensional porous structures useful for biomaterials and for scaffolds in tissue engineering. All-aqueous and organic solvent (hexafluoroisopropanol) modes of processing are compared relative to solution concentration of silk protein polymer and size of porogen (NaCl particles). The results clarify the range of conditions under which these biomaterial matrices can be formed, with a broader range of pore sizes and smoother surface morphology generated from the organic solvent process. These structures are directly applicable to fundamental studies of protein-based biomaterial assembly as well as cell interactions and tissue formation with these systems.

Fibrous proteins and tissue engineering

Fibrous proteins are finding broad impact in biomaterial systems for a range of cell and tissue studies. This impact derives from an improved insight into fundamental structure-function relationships, as well as the unique material properties attained with these protein polymers. Recent advances in the use of these protein systems in a variety of biomaterial and tissue engineering applications are reviewed, with a focus on approaches to control the structure, chemistry, and morphology of the biomaterials formed and enable cell and tissue outcomes to be directed in vitro and in vivo.