The cryopreservation of composite tissues: Principles and recent advancement on cryopreservation of different type of tissues

Cryopreservation of human cells and tissue has generated great interest in the scientific community since 1949, when the cryoprotective activity of glycerol was discovered. Nowadays, it is possible to reach the optimal conditions for the cryopreservation of a homogeneous cell population or a one cell-layer tissue with the preservation of a high pourcentage of the initial cells. Success is attained when there is a high recovery rate of cell structures and tissue components after thawing. It is more delicate to obtain cryopreservation of composite tissues and much more a whole organ. The present work deals with fundamental principles of the cryobiology of biological structures, with special attention to the transfer of liquids between intra and extracellular compartments and the initiation of the formation and aggregation of ice during freezing. The consequences of various physical and chemical reactions on biological tissue are described for different cryoprotective agents. Finally, we report a review of results on cyropreservation of various tissues, on the one hand, and various organs, on the other. We also report immunomodulation of antigenic responses to cryopreserved cells and organs.     

Regulation of Endochondral Cartilage Growth in the Developing Avian Limb: Cooperative Involvement of Perichondrium and Periosteum

The perichondrium and periosteum have recently been suggested to be involved in the regulation of limb growth, serving as potential sources of signaling molecules that are involved in chondrocyte proliferation, maturation, and hypertrophy. Previously, we observed that removal of the perichondrium and periosteum from tibiotarsi in organ culture resulted in an overall increase in longitudinal cartilage growth, suggesting negative regulation originating from these tissues. To determine if the perichondrium and periosteum regulate growth through the production of diffusible factors, we have tested various conditioned media from these tissues for the ability to modify cartilage growth in tibiotarsal organ cultures from which these tissues have been removed. Both negative and positive regulatory activities were detected. Negative regulation was observed with conditioned medium from (1) cell cultures of the region bordering both the perichondrium and the periosteum, (2) co-cultures of perichondrial and periosteal cells, and (3) a mixture of conditioned media from perichondrial cell cultures and periosteal cell cultures. The requirement for regulatory factors from both the perichondrium and periosteum suggests a novel mechanism of regulation. Positive regulation was observed with conditioned media from several cell types, with the most potent activity being from articular perichondrial cells and hypertrophic chondrocytes.     

The osteochondral ligament: a fibrous attachment between bone and articular cartilage in Rana catesbeiana

The anuran epiphyseal cartilage shows a lateral expansion that covers the external surface of the bone, besides other features that distinguish it from the corresponding avian and mammalian structures. The fibrous structure that attaches the lateral cartilage to the bone was characterized in this work. It was designated osteochondral ligament (OCL) and presented two main areas. There was an inner area that was closer to the periosteal bone and contained a layer of osteoblasts and elongated cells aligned to and interspersed with thin collagen fibers. The thin processes of the cells in this area showed strong alkaline phosphatase activity. The outer area, which was closer to the cartilage, was rich in blood vessels and contained a few cells amongst thick collagen fibers. TRITC-phaloidin staining showed the cells of the inner area to be rich in F-actin, and were observed to form a net around the cell nucleus and to fill the cell processes which extended between the collagen fibers. Cells of the outer area were poor in actin cytoskeleton, while those associated with the blood vessels showed intense staining. Tubulin-staining was weak, regardless of the OCL region. The main fibers of the extracellular matrix in the OCL extended obliquely upwards from the cartilage to the bone. The collagen fibers inserted into the bone matrix as Sharpey's fibers and became progressively thicker as they made their way through the outer area to the cartilage. Immunocytochemistry showed the presence of type I and type III collagen. Microfibrils were found around the cells and amongst the collagen fibrils. These microfibrils were composed of either type VI collagen or fibrilin, as shown by immunocytochemistry. The results presented in this paper show that the osteochondral ligament of Rana catesbeiana is a complex and specialized fibrous attachment which guarantees a strong and flexible anchorage of the lateral articular cartilage to the periosteal bone shaft, besides playing a role in bone growth.     

Preparation of adriamycin gelatin microsphere-loaded decellularized periosteum that is cytotoxic to human osteosarcoma cells

The purpose of this study was to develop a novel approach to treat bone osteosarcoma using a multipurpose scaffold aiming for local drug delivery. The slowly releasing microspheres was designed to deliver the chemotherapy drug adriamycin (ADM) and a decellularized (D) periosteum scaffold (which is known to be able to promote bone regeneration) was used to carry these microspheres. D-periosteum was obtained by physical and chemical decellularization. Histological results showed that the cellular components were effectively removed. The D-periosteum showed an excellent cytocompatibility and the ability to promote adhesion and growth of fibroblasts. Two kinds of slowly releasing microspheres, adriamycin gelatin microspheres (ADM-GMS) and adriamycin poly (dl-lactide-co-glycolide) gelatin microspheres (ADM-PLGA-GMS), were prepared and anchored to D-periosteum, resulting in two types of drug-releasing regenerative scaffolds. The effectiveness of these two scaffolds in killing human osteosarcoma cells was tested by evaluating cell viability overtime of the cancer cells cultured with the scaffolds. In summary, a gelatin/decellularized periosteum-based biologic scaffold material was designed aiming for local delivery of chemotherapy drugs for osteosarcoma, with the results showing ability of the scaffolds in sustaining release of the cancer drug and in suppressing growth of the cancer cells in vitro.     

The efficacy of non-surgical platelet-rich fibrin application on clinical periodontal parameters and periostin level in periodontitis: Clinical trial

Platelet-rich fibrin (PRF) has been widely used in regenerative dentistry due to many growth factors produced. Periostin, a matricellular protein, is a reliable marker for tissue regeneration. Periostin is part of the cellular matrix and regulates bone homeostasis. This study aims to explore the efficacy of PRF in improvement of the clinical periodontal parameters as an adjunct to the scaling and root planing and to evaluate periostin level in gingival crevicular fluid (GCF) at baseline, 1- and 3-month recall visits. Fourteen periodontitis patients who met the inclusion criteria were recruited in this study. Two contralateral periodontal pockets with 4–6 mm in depth in each patient were selected. The sites in every participant were randomly allocated into control sites or test sites. In control sites, only conventional scaling and root planing was carried out. In test sites, however, scaling and root planing method and PRF were applied. Periostin level in GCF and clinical periodontal parameters were measured. The test sites revealed greater relative attachment gain (2.614 ± 0.606 mm and 3.321 ± 0.668 mm) than control sites (1.285 ± 0.671 mm and 1.839 ± 0.632 mm) and a significant pocket reduction (2.535 ± 0.664 mm and 3.321 ± 0.668 mm) than the control sites (1.21 ± 0.508 mm and 1.892 ± 0.655 mm) at 1- and 3-month recall visits respectively. In the test sites, level of periostin (48.83 ± 9.3 ng/μl and 98.90 ± 24.94 ng/μl) were greater than periostin levels in the control sites (42.65 ± 7.03 ng/μl and 49.29 ± 15.14 ng/μl) at 1- and 3-month recall visits respectively. In conclusion, the non-surgical application of PRF as an adjunct to scaling and root planing significantly improved the clinical periodontal parameters through raising periostin level in GCF.     

Rat sinus mucosa- and periosteum-derived exosomes accelerate osteogenesis

Guided bone regeneration (GBR) is commonly used for alveolar bone augmentation. The paracrine mechanism in the field of bone tissue engineering has been emphasized in recent years and exosomes are considered to have the potential of promoting osteogenesis. We aimed to study the influence of sinus mucosa and periosteum on bone regeneration through paracrine stimulation, especially via exosomes, and compare the differences between them. Here, we report that conditioned medium (CM) from sinus mucosa-derived cells (SMCs) and periosteum-derived cells (PCs) and the isolated exosomes enhanced the proliferation, migration and osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BM-MSCs) in vitro. A rat model of femoral bone defects was used to demonstrate that the exosomes derived from SMCs (SMC-Exos) and PCs (PC-Exos) can accelerate bone formation in vivo. Furthermore, we present a preliminary discussion of the possible functional components involved in the effects of SMC-Exos and PC-Exos on bone regeneration. In conclusion, these results demonstrated that the sinus mucosa and periosteum can accelerate osteogenesis through paracrine effects and the exosomes play important roles in this process.