Prevention of Cartilage Degeneration in a Rat Model of Osteoarthritis by Intraarticular Treatment With Recombinant Lubricin

Carl R. Flannery, Richard Zollner, Chris Corcoran,Aled R. Jones, Adam Root,Moise´s A. Rivera-Bermu´dez, Tracey Blanchet, Jason P. Gleghorn, Lawrence J. Bonassar, Alison M. Bendele, Elisabeth A. Morris, and Sonya S. Glasson

ARTHRITIS & RHEUMATISM
Vol. 60, No. 3, March 2009, pp 840–847
DOI 10.1002/art.24304

Summary

Osteoarthritis is a degenerative disease characterized by the progressive loss of articular cartilage that affects the quality of life of millions of people around the world. Lubricants provide a crucial role in the protection of the articular cartilage by providing a friction-resistant coating to cartilage surfaces in joints. Lubricin is one of the glycoprotein in the lubricant that is proven to provide a significant role in preventing cartilage wear, synovial cell adhesion, synovial cell proliferation. Because mutations in the Lubricin gene are known to cause osteoarthritis (OA) symptoms, the paper reasoned that increase lubricin levels will be a good therapy for OA. Thus, the overall goal of the paper is to study the efficacy of injecting a novel recombinant of lubricin into rats with OA.

The generation of a novel recombinant of lubricin, instead of using the full gene sequence was done to optimize the production yields. The recombinant was created by exposing cDNA for human lubricin with restriction enzymes to replace the repetitive elements with a synthetic cassette (Figure 1a). The resulting construct was called LUB:1 and was transfected and purified from hamster ovary cells. LUB:1 protein was purified through affinity and size-exclusion chromatography, analyzed by SDS-PAGE gels, and are either stained with Coomassie blue (Lane 1 in Figure 1B) or transferred onto nitrocellulose membranes for Western blotting (Lane 2 in figure 1b)

Figure 1: A) a graphic representation of the relationship between Lubricin gene and the construct. B) Coomassie blue stains and Western Blotting after non-reducing SDS-PAGE. (Triangles indication the monomer and dimer of LUB:1. C) shows resistance of LUB:1 to digestion of neuraminidase (Land 3) and endo-O-glycosidase, but not both combined.


To test LUB:1 binding, bovine cartilages were extracted, native lubricin removed, specimens incubated with either LUB:1 or just PBS, and immunostained (Figure 2a). To test whether LUB:1 reduces friction, Bovine cartlage were exposed to either PBS alone or with increasing concentrations of LUB:1 and tested on a custom testing apparatus.

Figure 2: A) Bovine cartilage has an absence of lubricant binding. B) Arrows show the presence LUB:1 binding. C) Friction of coefficient is significantly lower with presence of LUB:1. D) Increase LUB:1 lowers cell adhesion



20 rat models had cartilage tears induced through surgery. One group of rats received doses of PBS. The other groups received intraarticular doses of LUB:1 either once a week or 3 times a week. Doses of albumin injections were used as a control for a group. After 4 weeks, rats were killed and OA was scored on cartilage degeneration, total joint score, width of severe lesions, significant cartilage degeneration width, bone score, and medial capsule width.

Figure 3: The analysis of histologic sections of the knee joints reveal score reductions throughout all categories with the presence of LUB:1, especially severe lesions width where there is a 83% decrease.





Figure 4: Rat joints with OA were treated with either PBS or LUB:1 3x a week. The figure shows that LUB:1 were able to preserve the cartilage structure, whereas the PBS-treated (4A) has an arrow indicating severe cartilage lesion.


Significance:
Since cartilage degeneration plays a major role in the progression of OA, studying therapies that preserve the structural integrity of the cartilage will lead to a reduction to a major cause of OA, resulting in an increase in mobility and quality of life in patients. Current treatments include anti-inflammatory drugs and inhibitors that provide symptomatic relief but do not prevent cartilage degeneration. Treatment strategies to inhibit collagenase have low efficacy and/or harmful side effects. This paper shows an novel approach to OA therapy using a higher yielding LUB:1 construct that produces significant results across most OA scores in rat models.