Tissue engineering for total meniscal substitution : Animal study in sheep model

Objective: The aim of the study was to investigate the use of a novel hyaluronic acid/polycaprolactone material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with expanded autologous chondrocytes. Two different surgical implantation techniques in a sheep model were evaluated. Methods: Twenty-four skeletally mature sheep were treated with total medial meniscus replacements, while two meniscectomies served as empty controls. The animals were divided into two groups: cell-free scaffold and scaffold seeded with autologous chondrocytes. Two different surgical techniques were compared: in 12 animals, the implant was sutured to the capsule and to the meniscal ligament; in the other 12 animals, also a transtibial fixation of the horns was used. The animals were euthanized after 4 months. The specimens were assessed by gross inspection and histology. Results: All implants showed excellent capsular ingrowth at the periphery. Macroscopically, no difference was observed between cell-seeded and cell-free groups. Better implant appearance and integrity was observed in the group without transosseous horns fixation. Using the latter implantation technique, lower joint degeneration was observed in the cell-seeded group with respect to cell-free implants. The histological analysis indicated cellular infiltration and vascularization throughout the implanted constructs. Cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. Conclusion: The current study supports the potential of a novel HYAFF/polycaprolactone scaffold for total meniscal substitution. Seeding of the scaffolds with autologous chondrocytes provides some benefit in the extent of fibrocartilaginous tissue repair

The Composition of Hyperacute Serum and Platelet-Rich Plasma Is Markedly Different despite the Similar Production Method

Autologous blood derived products, such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are widely applied in regenerative therapies, in contrast to the drawbacks in their application, mainly deriving from the preparation methods used. Eliminating the disadvantages of both PRP and PRF, hyperacute serum (HAS) opens a new path in autologous serum therapy showing similar or even improved regenerative potential at the same time. Despite the frequent experimental and clinical use of PRP and HAS, their protein composition has not been examined thoroughly yet. Thus, we investigated and compared the composition of HAS, serum, PRP and plasma products using citrate and EDTA by simple laboratory tests, and we compared the composition of HAS, serum, EDTA PRP and plasma by Proteome Profiler and ELISA assays. According to our results the natural ionic balance was upset in both EDTA and citrate PRP as well as in plasma. EDTA PRP contained significantly higher level of growth factors and cytokines, especially platelet derived angiogenic and inflammatory proteins, that can be explained by the significantly higher number of platelets in EDTA PRP. The composition analysis of blood derivatives revealed that although the preparation method of PRP and HAS were similar, the ionic and protein composition of HAS could be advantageous for cell function

Serum albumin coated bone allograft (BoneAlbumin) results in faster bone formation and mechanically stronger bone in aging rats

Serum albumin coated bone allografts (BoneAlbumin) have successfully supported bone regeneration in various experimental models by activating endogenous progenitors. However, the effect of tissue aging, linked to declining stem cell function, has yet to be explicitly examined within the context of BoneAlbuminꞋs regenerative capacity. Stem cell function was tested with an in vitro attachment assay, which showed that albumin coating increases stem cell attachment on demineralized bone surfaces in an aging cell population. Bone regeneration was investigated in vivo by creating critical size bone defects on the parietal bones of aging female rats. Demineralized bone matrices with and without serum albumin coating were used to fill the defects. Bone regeneration was determined by measuring the density and the size of the remaining bone defect with computed tomography. MicroCT and mechanical testing were performed on the parietal bone explants. In vivo CT and ex vivo microCT measurements showed better regeneration with albumin coated grafts. Additionally, the albumin coated group showed a two-fold increase in peak fracture force compared to uncoated allografts. In the present study, serum albumin coated demineralized bone matrices successfully supported faster and functionally superior bone regeneration in aging rats. Since stem cell function, a key contributor of bone remodeling, decreases with age and serum albumin is an effective activator of endogenous progenitor cells, this method could be an effective and safe adjuvant in bone regeneration of aging adult and osteo-compromised populations

A protocol for gene expression analysis of chondrocytes from bovine osteochondral plugs used for biotribological applications

RNA isolation from human or animal cartilage tissue is necessary when performing mechanical or biotribological applications. Despite no influence on the cells and no alterations in gene expression patterns, enzymatic digestion of tissues should be avoided as it’s known that the expression of collagen 2 can be effected (Hayman et al., 2006 [1]). After mechanical or biotribological tests alternative options with an immediate disruption of the tissue should be contemplated. To obtain RNA, different tissue homogenization and disruption methods are available on the market (Yu et al., 2004 [2]), but not everyone is suitable for cartilage. Some of them neither homogenize the cartilage, while others are producing a lot of foam during disruption process. After trying some of the currently available methods, we chose the MagNA Lyser Instrument from Roche to disrupt the cartilage and further isolate RNA by using the Fibrous Tissue Kit from Qiagen. After RNA isolation, cDNA synthesis was performed by additionally adding RNA from bacteriophage MS2 for stabilization purposes. For the RTqPCR bovine primers were designed and tested for efficiency to confirm that the whole gene expression analysis is working. Our protocol explains a whole method to perform gene expression analysis from bovine cartilage, but can also be used for human or any other animal tissue