&NA; This study evaluates the feasibility of growing tissue‐engineered cartilage in the shape of a human ear using chondrocytes seeded onto a synthetic biodegradable polymer fashioned in the shape of a 3‐year‐old child's auricle. A polymer template was formed in the shape of a human auricle using a nonwoven mesh of polyglycolic acid molded after being immersed in a 1% solution of polylactic acid. Each polyglycolic acid‐polylactic acid template was seeded with chondrocytes isolated from bovine articular cartilage and then implanted into subcutaneous pockets on the dorsa of 10 athymic mice. The three‐dimensional structure was well maintained after removal of an external stent that had been applied for 4 weeks. Specimens harvested 12 weeks after implantation and subjected to gross morphologic and histologic analysis demonstrated new cartilage formation. The overall geometry of the experimental specimens closely resembled the complex structure of the child's auricle. These findings demonstrate that polyglycolic acid‐polylactic acid constructs can be fabricated in a very intricate configuration and seeded with chondrocytes to generate new cartilage that would be useful in plastic and reconstructive surgery. (Plast. Reconstr. Surg. 100: 297, 1997.)

Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear

Osteochondral lesions due to injury or other pathology commonly result in the development of osteoarthritis and progressive joint destruction. Bioengineered scaffolds are widely studied for regenerative surgery strategies in osteochondral defect management, also combining the use of stem cells, growth factors and hormones. The utility in tissue engineering of human adipose-derived stem cells (ASCs) isolated from adipose tissue has been widely noted. Autologous platelet-rich plasma (PRP) represents an alternative strategy in regenerative medicine for the local release of endogenous growth factors and hormones. Here we compared the effects of three-dimensional (3D) collagen type I scaffold culture and combined treatment with PRP and human recombinant insulin on the chondro-/osteogenic differentiation of ASCs. Histochemical and biomolecular analyses demonstrated that chondro-/osteogenic differentiation was increased in ASC-populated 3D collagen scaffolds compared with two-dimensional (2D) plastic dish culture. Chondro-/osteogenic differentiation was further enhanced in the presence of combined PRP (5% v/v) and insulin (100 nm) treatment. In addition, chondro-/osteogenic differentiation associated with the contraction of ASC-populated 3D collagen scaffold and increased β1/β3-integrin expression. Inhibition studies demonstrated that PRP/insulin-induced chondro-/osteogenic differentiation is independent of insulin-like growth factor 1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) signalling; IGF-R1/mTOR inhibition even enhanced ASC chondro-/osteogenic differentiation. Our findings underline that 3D collagen scaffold culture in association with platelet-derived growth factors and insulin favour the chondro-/osteogenic differentiation of ASCs, suggesting new translational applications in regenerative medicine for the management of osteochondral defects. Copyright © 2016 John Wiley & Sons, Ltd.

Combined treatment with platelet‐rich plasma and insulin favours chondrogenic and osteogenic differentiation of human adipose‐derived stem cells in three‐dimensional collagen scaffolds

Fracture healing encompasses a succession of dynamic multifactorial metabolic events, which ultimately re-establishes the integrity of the biomechanical properties of the bone. Up to 10% of the fractures occurring annually will need additional surgical procedures because of impaired healing. The aim of this article is to review the current literature regarding the use of bone marrow aspirate concentrate (BMAC) and its effectiveness in the management of bone defects. We have included all published clinical literature investigating the development, techniques and applications of BMAC. Language, design and risk of bias did not deter the initial inclusion of any study. Our search was exclusively limited to studies involving human subjects. A PRISMA compliant search was carried out as published in 2009. This included the online databases: PubMed, EMBASE, clinical trial.gov
 and the Cochrane library from 1960 to the end of May 2015. MeSH terms used included: “Bone” AND “Marrow” AND “Aspirate” AND “Concentrate” AND “Bone Defects” AND “NONUNION”. Eligible studies were independently appraised by two authors using the Critical Appraisal Skills Program checklist. For the purpose of narrative review, relevant studies were included irrespective of methodology or level of evidence. Thirty-four of the 103 (48 PubMed and 55 EMBASE) results yielded by the preliminary search were included. Exclusions included three duplicate records, six letters, 17 non-orthopaedics related studies and four records irrelevant to our search topic. The CASP appraisal confirmed a satisfactory standard of 31 studies. They all had clearly defined objectives, were well designed and conducted appropriately to meet them. The published studies reported the use of BMAC in non-union and fracture healing (15 studies), bone defects (nine studies), spine fusion (two studies), distraction osteogensis (two studies) and complications related to the use of BMAC (seven studies). Stem cells found in BMAC have the potential to self-renew, undertake clonal expansion and differentiate into different musculoskeletal tissues. The commercial processing of BMAC needs to be optimized in order to achieve a consistent end product, which will provide predicable and translatable results. The future potential of cell characterization in order to determine the optimum cell for repair/regeneration of bone also needs to be explored. Level of Evidence: Systematic Review of minimum level IV studies.

A systematic review of the clinical applications and complications of bone marrow aspirate concentrate in management of bone defects and nonunions.

There has been great interest in bone marrow aspirate concentrate (BMAC) as a cost effective method in delivering mesenchymal stem cells (MSCs) to aid in the repair and regeneration of cartilage defects. Alongside MSCs, BMAC contains a range of growth factors and cytokines to support cell growth following injury. However, there is paucity of information relating to the basic science underlying BMAC and its exact biological role in supporting the growth and regeneration of chondrocytes. The focus of this review is the basic science underlying BMAC in relation to chondral damage and regeneration.

The basic science of bone marrow aspirate concentrate in chondral injuries

Fracture healing is a complex process regulated by a variety of cells and signalling molecules which act both locally and systemically. The aim of this study was to investigate potential changes in patients’ mesenchymal stem cells (MSCs) in the iliac crest (IC) bone marrow (BM) and in peripheral blood (PB) in relation to the severity of trauma and to correlate them with systemic changes reflective of inflammatory and platelet responses following fracture. ICBM samples were aspirated from two trauma groups: isolated trauma and polytrauma (n = 8 and 18, respectively) at two time-points post-fracture and from non-trauma controls (n = 7). Matched PB was collected every other day for a minimum of 14 days. BM MSCs were enumerated using colony forming-fibroblast (CFU-F) assay and flow cytometry for the CD45-CD271+ phenotype. Regardless of the severity of trauma, no significant increase or decrease in BM MSCs was observed following fracture and MSCs were not mobilised into PB. However, direct positive correlations were observed between changes in the numbers of aspirated BM MSCs and time-matched changes in their serum PDGF-AA and -BB. In vitro, patients’ serum induced MSC proliferation in a manner reflecting changes in PDGFs. PDGF receptors CD140a and CD140b were expressed on native CD45-CD271+ BM MSCs (average 12% and 64%, respectively) and changed over time in direct relationship with platelets/PDGFs. Platelet lysates and other platelet-derived products are used to expand MSCs ex vivo. This study demonstrates that endogenous PDGFs can influence MSC responses in vivo. This indicates a highly dynamic, rather than static, MSC nature in humans.

/pdf/the-systemic-influence-of-platelet-derived-growth-factors-on-1a9kl3qet3.pdf

The systemic influence of platelet-derived growth factors on bone marrow mesenchymal stem cells in fracture patients

Cartilage defect caused by disease or trauma remains a challenge for surgeons, owning to the limited healing capacity of cartilage tissues. Cartilage tissue engineering provides a novel approach to address this issue, and appears promising for patients with cartilage defects. The cell scaffold, as one of the three key elements of tissue engineering, plays an important role in cartilage tissue engineering. Platelet‑rich plasma (PRP), which is a fraction of the plasma containing multiple growth factors, has become a major research focus in the context of its use as a bioactive scaffold for tissue engineering. Therefore, we investigated the value of using PRP scaffolds combined with chondrocytes in cartilage tissue engineering. In this study, we examined the levels of growth factors in PRP, and the effects of PRP on cell proliferation and matrix synthesis in rabbit chondrocytes cultured in PRP. Short-term in vitro culture followed by long‑term in vivo implantation was performed to evaluate the chondrogenesis of neocartilage in vivo. The results show that PRP may provide a suitable environment for the proliferation and maturation of chondrocytes, and can be used as a promising bioactive scaffold for cartilage regeneration.

/pdf/the-application-of-autologous-platelet-rich-plasma-gel-in-rf1ncaaun2.pdf

The application of autologous platelet‑rich plasma gel in cartilage regeneration

The objective of this study was to investigate the efficacy of local injection of botulinum toxin A for treating axillary osmidrosis. One hundred and fifty patients with axillary osmidrosis were randomly divided to receive botulinum toxin A injection treatment (50 U of botulinum toxin A was injected intracutaneously into 6-20 different sites within each axilla, n = 74) or surgical excision of the apocrine glands (n = 76). The patients were followed up for 1-3 months to analyze the therapeutic effect and complications of the two methods. The curative effect in patients with mild and moderate axillary osmidrosis was not significantly different between the botulinum toxin A injection group and operation group. However, for patients with severe axillary osmidrosis, surgery treatment seemed to be superior to botulinum toxin A treatment (P = 0.005). There was also no significant difference in the modified Dermatology Life Quality Index between the two treatments. Two cases showed complications related to hemorrhage and incision infection in the operation group. In conclusion, local injection of botulinum toxin A is a safe, fast and effective treatment for mild and moderate axillary osmidrosis, but the long-term effect remains to be further investigated.

Local injection of botulinum toxin A: an alternative therapy for axillary osmidrosis.

Cartilage tissue engineering provides a new method in the treatment of cartilage defects, and adipose derived stem cells seem to be an ideal seed cell in cartilage tissue engineering because of its characteristics. However, ossification after in vivo implantation of tissue engineered cartilage remains a challenge. Thrombospondin-1 which has been reported to have an inhibitory effect on angiogenesis, may play an important role in inhibiting the ossification of tissue engineered cartilage constructed by adipose derived stem cells. Therefore, the effect of thrombospondin-1 in inhibiting the ossification of tissue engineered cartilage was evaluated in this study. Lentivirus vectors carrying thrombospondin-1 cDNA were transfected into adipose derived stem cells, and the transfected cells were used in the experiments. The expression of thrombospondin-1 was evaluated by quantitative reverse transcriptase-polymerase chain reaction and western blot, and the effects of thrombospondin-1 over-expression on angiogenesis were analyzed by angiogenesis assays. The quality of tissue engineered cartilage and the degree of ossification were assessed by biomechanical and molecular biology methods. The results showed that thrombospondin-1 infected cells have a high expression of thrombospondin-1 in mRNA and protein level, which inhibited the tube formation of endothelial cells, indicating the anti-angiogenic effects. Gene expression analyses in vitro showed that thrombospondin-1 inhibits the osteogenic differentiation of adipose derived stem cells significantly, and the results of in vivo study revealed that thrombospondin-1 significantly inhibits the expression of osteogenic genes. Compared to that in the control group, tissue engineered cartilage constructed by thrombospondin-1 transfected adipose derived stem cells in vivo showed a higher GAG content and lower compressive modulus, which indicating lower level of ossification. In conclusion, the current study indicated that the anti-angiogenic factor thrombospondin-1 suppresses the osteogenic differentiation of adipose derived stem cells in vitro, and inhibits ossification of tissue engineered cartilage constructed by adipose derived stem cells in vivo.

Thrombospondin-1 inhibits ossification of tissue engineered cartilage constructed by ADSCs.

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The application of autologous platelet‑rich plasma gel in cartilage regeneration

Local injection of botulinum toxin A: an alternative therapy for axillary osmidrosis.

Thrombospondin-1 inhibits ossification of tissue engineered cartilage constructed by ADSCs.