Antimicrobial Effectiveness Testing of Resorbable Electrospun Fiber Matrix per United States Pharmacopeia (USP) <51>.

Contamination of surgical, traumatic, and chronic wounds with microorganisms presents a challenge to successful wound healing. In the present in vitro study, a synthetic electrospun fiber matrix (SEFM) cleared for use in the management of chronic, surgical, and traumatic wounds underwent USP (United States Pharmacopeia) <51> Antimicrobial Effectiveness Testing to determine its in vitro effectiveness against various microorganisms commonly found in non-healing wounds. The SEFM was tested in both sheet (s-SEFM) and micronized form (m-SEFM) against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus brasiliensis, Candida albicans, Proteus mirabilis, and Enterococcus faecalis. Testing was performed per the USP <51> standard on days 7, 14, and 28. Both the s-SEFM and m-SEFM met the USP <51> acceptance criteria for all microorganisms. The results obtained for s-SEFM demonstrated >1-log10 reduction against E. coli, S. aureus, P. aeruginosa, P. mirabilis, E. faecalis, and C. albicans at day 7; >3-log10 reduction with no detection of these microbes at days 14 and 28, and no increase from initial inoculum at days 7, 14, and 28 against A. brasiliensis. The results obtained for m-SEFM demonstrated >3-log10 reduction with no detectable microorganisms at day 7. The results observed in this study indicate that the SEFM is effective in vitro at inhibiting bacterial and fungal growth and colonization per USP <51> testing.

Clinical Application of Bioresorbable, Synthetic, Electrospun Matrix in Wound Healing.

Electrospun polymeric matrices have long been investigated as constructs for use in regenerative medicine, yet relatively few have been commercialized for human clinical use. In 2017, a novel electrospun matrix, composed of two synthetic biocompatible polymers, polyglactin 910 (PLGA 10:90) and polydioxanone (PDO) of varying pore and fiber sizes (i.e., hybrid-scale) was developed and cleared by the FDA for human clinical use. The present review aims to explain the mechanism of action and review the preclinical and clinical results to summarize the efficacy of the matrix across multiple use cases within the wound care setting, including an assessment of over 150 wounds of varying etiologies treated with the synthetic matrix. Clinical data demonstrated effective use of the synthetic hybrid-scale fiber matrix across a variety of wound etiologies, including diabetic foot and venous leg ulcers, pressure ulcers, burns, and surgical wounds. This review represents a comprehensive clinical demonstration of a synthetic, electrospun, hybrid-scale matrix and illustrates its value and versatility across multiple wound etiologies.

Tissue-engineered cartilaginous constructs for the treatment of caprine cartilage defects, including distribution of laminin and type IV collagen.

The purpose of this study was the immunohistochemical evaluation of (1) cartilage tissue-engineered constructs; and (2) the tissue filling cartilage defects in a goat model into which the constructs were implanted, particularly for the presence of the basement membrane molecules, laminin and type IV collagen. Basement membrane molecules are localized to the pericellular matrix in normal adult articular cartilage, but have not been examined in tissue-engineered constructs cultured in vitro or in tissue filling cartilage defects into which the constructs were implanted. Cartilaginous constructs were engineered in vitro using caprine chondrocyte-seeded type II collagen scaffolds. Autologous constructs were implanted into 4-mm-diameter defects created to the tidemark in the trochlear groove in the knee joints of skeletally mature goats. Eight weeks after implantation, the animals were sacrificed. Constructs underwent immunohistochemical and histomorphometric evaluation. Widespread staining for the two basement membrane molecules was observed throughout the extracellular matrix of in vitro and in vivo samples in a distribution unlike that previously reported for cartilage. At sacrifice, 70% of the defect site was filled with reparative tissue, which consisted largely of fibrous tissue and some fibrocartilage, with over 70% of the reparative tissue bonded to the adjacent host tissue. A novel finding of this study was the observation of laminin and type IV collagen in in vitro engineered cartilaginous constructs and in vivo cartilage repair samples from defects into which the constructs were implanted, as well as in normal caprine articular cartilage. Future work is needed to elucidate the role of basement membrane molecules during cartilage repair and regeneration.

Engineering endostatin-expressing cartilaginous constructs using injectable biopolymer hydrogels.

The release of an anti-angiogenic agent, such as type XVIII/endostatin, from an implantable scaffold may be of benefit in the repair of articular cartilage. The objectives of this study are to develop an injectable mesenchymal stem cell (MSC)-incorporating collagen-based hydrogel capable of undergoing covalent cross-linking in vivo and overexpressing endostatin using nonviral transfection, and to investigate methods for the retention of the endostatin protein within the scaffolds. The effects of different cross-linking agents (genipin, transglutaminase-2, and microbial transglutaminase) and different binding molecules for endostatin retention (heparin, heparan sulfate, and chondroitin sulfate) are evaluated. Cartilaginous constructs that overexpress endostatin for 3 weeks are successfully engineered. Most of the endostatin is released into the surrounding media and is not retained within the constructs. The presence of two common basement membrane molecules, laminin and type IV collagen, which have been reported in developing and mature articular cartilage and are generally associated with type XVIII collagen in vivo, is also observed in the engineered cartilaginous constructs. Endostatin-producing cartilaginous constructs can be formulated by growing nonvirally transfected mesenchymal stem cells in collagen gels covalently cross-linked using genipin, transglutaminase-2, and microbial transglutaminase. These constructs warrant further investigation for cartilage repair procedures. The novel finding of laminin and type IV collagen in the engineered cartilage constructs may be of importance for future work toward understanding the role of basement membrane molecules in chondrogenesis and in the physiology and pathology of articular cartilage.

The Biological Response following Autogenous Bone Grafting for Large-Volume Defects of the Knee: Index Surgery through 12 to 21 Years' Follow-up.

OBJECTIVE: This report focuses on the biological events occurring at various intervals following autogenous bone grafting of large-volume defects of the knee joint's femoral condyle secondary to osteochondritis dissecans (OCD) or osteonecrosis (ON). It was hypothesized that the autogenous bone graft would integrate and the portion exposed to the articular surface would form fibrocartilage, which would endure for years. METHODS: Between September 29, 1987 and August 8, 1994, there were 51 patients treated with autogenous bone grafting for large-volume osteochondral defects. Twenty-five of the 51 patients were available for long-term follow-up up to 21 years. Patient follow-up was accomplished by clinical opportunity and intentional research. Videotapes were available on all index surgeries for review and comparison. All had preoperative and postoperative plain film radiographs. Long-term follow-up included MRI up to 21 years. Second-look arthroscopy and biopsy were obtained on 14 patients between 8 weeks and 20 years. RESULTS: Radiological assessment showed the autogenous bone grafts integrated with the host bone. The grafts retained the physical geometry of the original placement. MRI showed soft tissue covering the grafts in all cases at long-term follow-up. Interval biopsy showed the surface covered with fibrous tissue at 8 weeks and subsequently converted to fibrocartilage with hyaline cartilage at 20 years. CONCLUSION: Autogenous bone grafting provides a matrix for large osteochondral defects that integrates with the host bone and results in a surface repair of fibrocartilage and hyaline cartilage that can endure for up to 20 years.

Engineering endostatin-producing cartilaginous constructs for cartilage repair using nonviral transfection of chondrocyte-seeded and mesenchymal-stem-cell-seeded collagen scaffolds.

Although there is widespread recognition of the importance of angiogenesis in tissue repair, there is little work on the inhibition of angiogenesis in the context of tissue engineering of naturally avascular tissues, like articular cartilage. The objective was to engineer a collagen-scaffold-based cartilaginous construct overexpressing a potent antiangiogenic factor, endostatin, using nonviral transfection. Endostatin-plasmid-supplemented collagen scaffolds were seeded with mesenchymal stem cells and chondrocytes and cultured for 20–22 days. The effects of the following variables on endostatin expression and chondrogenesis were examined: collagen scaffold material, method of nonviral vector incorporation, plasmid load, culture medium, and oxygen tension. An increase and peak of endostatin protein was observed during the first week of culture, followed by a decrease to low levels, suggesting that overexpression of endostatin could be sustained for several days using the nonviral vector. The amount of endostatin produced was tunable with the external factors. Chondrogenesis was observed in the engineered constructs cultured in chondrogenic medium at the 3-week time point, demonstrating that endostatin did not inhibit the chondrogenic potential of mesenchymal stem cells or the general viability of the cells. The ability to engineer endostatin-expressing cartilaginous constructs will be of value for future work exercising regulatory control of angiogenesis in cartilage repair.

Non-viral endostatin plasmid transfection of mesenchymal stem cells via collagen scaffolds.

Angiogenesis is critical in the early stage of reparative processes and tissue regeneration, but the persistence of a vascular network may interfere with later transformation/maturation in naturally avascular tissues such as articular cartilage. Our supposition is that the timed delivery of an anti-angiogenic factor in cartilage tissue engineering may facilitate the formation of hyaline cartilage by inducing the regression of vascularization. To this end our overall goal is to prepare an off-the-shelf scaffold containing the gene for a potent anti-angiogenic factor. The objective of this study was to investigate the use of a type I/III collagen scaffold for the non-viral transfection of marrow stromal cells (MSCs, also referred to as mesenchymal stem cells) with the plasmid encoding endostatin. Caprine MSCs were transfected by the naked plasmid alone and plasmid incorporated into a cationic lipid complex in three experiments: 1) cells were transfected in monolayer; 2) monolayer-transfected cells were grown in a collagen sponge-like scaffold; and 3) non-transfected cells were grown in a collagen scaffold containing the naked plasmid and endostatin lipoplex. Independent variables were the passage number of the cells and the plasmid loading. The amount of endostatin released by the cells into the medium was measured using an ELISA. The results demonstrated the overexpression of endostatin by MSCs growing in the endostatin lipoplex-supplemented collagen scaffolds. Endostatin released by the cell-seeded scaffolds reached a peak of 13ng/ml for scaffolds incorporating as little as 20mug of plasmid, at the 3-day collection period ending 5 days post-seeding. The accumulated endostatin synthesis over a 2-week period began to achieve what may be a therapeutic level. MSCs transfected with the endostatin gene in monolayer continued to express the gene when grown in the collagen scaffolds. The results demonstrate the promise of the non-viral delivery of the gene for this potent anti-angiogenic protein to MSCs via a collagen scaffold.

Nocturnal hypoglycemia detected with the Continuous Glucose Monitoring System in pediatric patients with type 1 diabetes.

OBJECTIVE: To use the Continuous Glucose Monitoring System (CGMS, MiniMed, Sylmar, Calif) to determine if bedtime blood glucose levels were associated with the occurrence of nocturnal hypoglycemia. STUDY DESIGN: Patients (n = 47, 18 boys, mean age 11.8 +/- 4.6 years) with type 1 diabetes used CGMS for 167 nights. Data were analyzed for glucose 100 mg/dL and 150 mg/dL. RESULTS: A glucose value of 100 mg/dL, P = NS), and no bedtime glucose value between 110 and 300 mg/dL decreased the incidence of nocturnal hypoglycemia to