gms | German Medical Science

Objectives: Subchondral bone is involved in a variety of diseases affecting both the articular cartilage and bone. Although some studies have shown advances in repairing cartilage defects, regenerative treatment options for osteochondral defects are not solved. Moreover, also matrix associated cartilage-based strategies require solid fixation of graft/scaffold composites at the osseous transplantation site. In this study we investigated the tissue regenerative capacity of a one-step procedure using vacuum-impregnated collagen membranes with embedded beta-TCP granules.

Methods: Porcine collagen I/III scaffolds (Chondro-Gide, GC) and beta-TCP granules embedded into a collagen I sponge (Cerasorb Foram CC) were loaded with chondral and subchondral fragments respectively, harvested in 18 patients qualified for total knee arthroplasty. For this purpose, cartilage specimens were treated by a 3.8 arthroscopic shaver and coated onto the scaffolds under vacuum using an innovative tissue collector (ChondroFlo). Afterwards, scaffolds were cultured for 8 weeks. Adhesion and distribution of tissue fragments were investigated. Cellular viability and proliferation are analyzed by MTT staining, ATP quantification, and population doubling time in the control group. The differentiation is evaluated based on mineralization processes through photometric measurement of Alkaline Phosphatase, Alizarin Red staining in the osteogenic control group, and 3-D Micromass cultivation with subsequent Alcian Blue staining in the chondrogenic control group. Differentiation of the cells on the scaffolds is analyzed through RNA extraction and PCR. Cell morphology is assessed through Scanning Electron Microscopy (SEM) and Confocal Scanning Laser Microscopy (CLSM).

Results and conclusion: The average surface area of cultivated minced tissue is 158,000 µm² in chondrogenic fragments and 57,500 µm² in osteogenic fragments. After in-vitro cultivation of 5 weeks, viable cells could be detected on both scaffolds through MTT staining. The ATP quantification suggests a higher cellular activity on CC (85,149 nM) than on GC (240 nM) which is confirmed through an increased growth rate in the osteogenic cell culture (6.7 d) as opposed to the chondrogenic counterpart (7.5 d) in the in-vitro control group. The cellular outgrowth on GC with autologous chondrocytes as control shows the markers ACAN and SOX9 indicating chondrogenic differentiation. The CC with a pHOB cell line as control shows gene expression markers osteocalcin, bone sialo protein, Runx, and osteoprotegerin indicating osteogenic differentiation. Cell morphology by SEM and CLSM present cells ranging from a polygonal to an elongated appearance with thin filopodia were visible on CC. On GC, the cells appear to be dedifferentiated, showing a slender morphology rather than the usual spheroidal shape of chondrocytes, possibly related to the cultivation duration.

Conclusion: Vacuum impregnation allow successful coating of collagen scaffolds with tissue fragments.