Propionibacterium acnes infection of the shoulder after arthroplasty is a common complication. Current detection methodologies for P. acnes involve prolonged anaerobic cultures that can take up to three weeks before findings can be reported. Our aim was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach that is both sensitive and specific to P. acnes that would enable a 24-hour turnaround between biopsy and results.

Comparisons between the 16S ribosomal sequences of P. acnes and closely related bacteria identified two unique regions in P.acnes to which PCR primers were designed. Additionally, two unique restriction enzyme cut sites for HaeIII were identified within this amplicon. To test the PCR method, arthroscopic surgical biopsies were mechanically homogenised and boiled for 20 minutes to lyse the cellular membranes. PCR was performed using standard conditions followed by a one hour HaeIII enzymatic digest of the PCR product. Resultant fragments were visualised on polyacrylamide gels stained with ethidium bromide. All experiments included no-template controls to rule out reagent contamination and independently confirmed P. acnes DNA as a positive control. Serial dilutions of P. acnes cultures in Robertson's cooked-meat broth and spectrophotometric analysis of cellular concentration were used to assess the sensitivity of the PCR reaction.

A unique 564 base-pair PCR amplicon was derived from different strains of P. acnes. This amplicon was confirmed as P. acnes DNA by gel excision and DNA sequencing. HaeIII digests of the amplicon yielded 3 restriction fragments at the sizes predicted by in silico analyses. Sensitivity testing confirmed that as few as 10 P. acnes cells in a 50µl reaction volume could be detected using this assay. P. acnes was also detected in surgical biopsy samples.

P. acnes infections following shoulder arthroplasty are a serious complication placing a burden on the healthcare system and the patient due to the lengthy surgical revision process that follows. The infections are also difficult to diagnose. This unique assay combines the sensitivity of PCR with the specificity of RFLP mapping to specifically identify P. acnes in surgical isolates. We anticipate that this assay will allow us to determine if a biopsy is P. acnes positive within 24-hours of sampling, allowing for more aggressive antibiotic therapy and monitoring to avoid implant failure and revision surgery. Additionally, this PCR-RFLP method may decrease the false positive rate of extended length cultures due to P. acnes contamination.

Rotator cuff repair is performed to treat shoulder pain and disability. Failure of the tendon repair site is common; one strategy to improve healing is to enforce a period of post-operative immobilisation. Immobilisation may have unintended effects on tendon healing. Tenocytes under uniaxial strain form more organised collagen and up regulate expression of proliferative genes. Vitamin C (ascorbic acid), an anti-oxidant that is a co-factor for collagen synthesis, has also been reported to enhance collagen deposition and organisation. The purpose of this study was to compare human tenocyte cultures exposed to uniaxial cyclical strain with or without slow-release ascorbic acid (ascorbyl-2 phosphate) to determine their individual and combined effects on tissue remodelling and expression of tissue repair genes.

Rotator cuff tissues were collected from degenerative supraspinatus tears from eight patients. Tenocytes were incorporated into 3D type I collagen culture matrices. Cultures were divided into four groups: 1) ascorbic acid (0.6mMol/L) + strain (1%–20% uniaxial cyclic strain at 0.1 Hz), 2) ascorbic acid unstrained, 3) strain + vehicle 4) unstrained + vehicle. Samples were fixed in paraffin, stained with picrosirius red and analysed with circular polarising light. A second set of cultures were divided into three groups: 1) 0.5mM ascorbic acid, 2) 1mM ascorbic acid, 3) vehicle cultured for 24, 72, 120 and 168 hours. Cell-free collagen matrix was used as a control. Tenocyte proliferation was assessed using the water soluble tetrazolium-1 (WST1) assay and f tissue repair gene expression (TGFB1, COL1A1, FN1, COLIII, IGF2, MMP1, and MMP13), were analysed by qPCR. The data were analysed using a Split model ANOVA with contrast and bonferroni correction and a one-way ANOVAs and Tukey's test (p<0.05 was significant).

Our results indicated that unstrained cultures with or without exposure to slow release ascorbic acid exhibited greater picrosirius red birifringency and an increase in collagen fiber deposition in a longitudinal orientation compared to strained tenocytes. We found that slow release ascorbic acid promoted significant dose and culture-time dependent increases in tenocyte proliferation (p<0.05) but no obvious enhancement in collagen deposition was evident over cultures without ascorbic acid supplementation.

Based on these data, applying strain to tenocytes may result in less organised formation of collagen fibers, suggestive of fibrotic tissue, rather than tendon remodelling. This may indicate that a short period of immobilisation post-rotator cuff repair is beneficial for the healing of tendons. Exposure to slow release ascorbic acid enhanced tenocyte proliferation, suggesting that supplementation with Vitamin C may improve tendon repair post-injury or repair. Future studies will assess levels of tissue repair-associated proteins as well as comparing traumatic and degenerative rotator cuff tears to healthy uninjured rotator cuff tissue.