Injectable enzymatically cross linkable hydrogels: a minimally invasive cell free approach to regenerate chondral defects

S. Both, S. de Cockelaere, R. Wang, N. Vos, G. Li, Y. Lin, R. van Weeren, P. Dijkstra, M. Karperien

Research output: Contribution to journalMeeting AbstractOther research output


Purpose: Focal cartilage defects as a consequence of trauma are a major risk factor for the development of early onset osteoarthritis. These defects still pose a largely unresolved problem for the treating physician. Previously, we have developed an injectable in situ gelating hydrogel that can be applied in an arthroscopic procedure to fill up cartilage defects by simple injection. These hydrogels consist of hyaluronic acid - tyramine and dextran - tyramine conjugates that cross link in in a cell-friendly enzymatic, peroxidase-based reaction, initiated by non-toxic concentrations of H2O2. During the cross linking reaction the hydrogels co-valently attach to the cartilage resulting in strong bonding and fixation of the hydrogel in the defect. These hydrogels possess chemoattractant properties facilitating the ingrowth of cells as demonstrated in an ex vivo chondral plug model opening the possibility for cell-free cartilage repair. The aim of this study is to test the use of these injectable hydrogels for cartilage repair in an orthotopic chondral defect rabbit model side-by-side compared with autologous chondrocyte implantation. In addition, we evaluated the concept in an equine model for focal cartilage defects.

Methods: Three male rabbits were sacrificed to establish cultures of primary human chondrocytes for implantation purposes. In a pilot rabbit experiment skeletally mature female rabbits were operated under anesthesia and two 4mm wide chondral defects were created in each knee joint. The defects were left untreated, filled up with hydrogel only, or with hydrogel prior mixed with chondrocytes. The various combinations of hydrogel precursors were injected in a liquid state in the defect and left to settle in a mild enzymatically mediated cross linking reaction which took place within less than 20 seconds. Rabbits were sacrificed 4 weeks and 10 weeks after treatment and tissues were collected for histology. In a pilot experiment two horses were operated under general anaesthesia in a fully arthroscopic procedure. In each knee joint, 5mm wide chondral defects were created. These defects were in the same arthroscopic procedure completely filled with the hydrogel. Synovial fluid was collected after 1, 2, 3, 5, 7 and 14 days after surgery. After two weeks horses were humanely euthanisized and tissue was processed for histology.

Results: In pilot experiments in rabbits, chondral defects were completely repaired using the injectable hydrogels after 10 weeks of surgery. Cell-free hydrogels appeared as efficient as cell-containing hydrogels. The data are now confirmed in a larger study group in which treatment with hydrogels is compared to microfracture. In the equine model we demonstrated that the injectable hydrogels could be used to fill up focal chondral defects in an completely arthroscopic procedure. Synovial fluid sampling demonstrated a clinically not relevant small increase in white blood cell count and protein count in the first 2 days after surgery which returned to base-line after 3 to 5 days. Clinical examination and follow up of the operated joints demonstrated normal response to arthroscopic surgery: no adverse effects were noted demonstrating the safety of the procedure. The horses were able to make functional use of their treated legs within a few days and walked normally 2-weeks after surgery. At this time point visual inspection demonstrated the presence of hydrogels in each of the defects. Histological examination demonstrated the presence of cell layers on top of the hydrogel and invasion of cells into the hydrogel both from the top and the bottom. The invading cells were organized in columns, like in normal cartilage, and stained positive for typical chondrocyte markers. They actively deposited glycosaminoglycans.

Conclusions: This study demonstrates the feasibility of developing an arthroscopic and completely cell-free treatment of chondral defects. It also demonstrates the presence of populations of migratory cells in the traumatized joint. These cells can actively migrate to and invade an appropriate scaffolding material in vivo and start the deposition of cartilage matrix. In the future, this work may translate into a biomaterial based regenerative treatment of osteoarthritis by harnessing the regenerative potential of these migratory cells.
Original languageEnglish
Pages (from-to)S161-S161
Number of pages1
JournalOsteoarthritis and cartilage
Issue numberSuppl. 1
Publication statusPublished - 2016
Event2016 OARSI World Congress on Osteoarthrtis - Amsterdam RAI, Amsterdam, Netherlands
Duration: 31 Mar 20163 Apr 2016


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