Large cartilage lesions in younger patients can be treated by fresh osteochondral allograft transplantation, a surgical technique that relies on stable initial fixation and a minimum chondrocyte viability of 70% in the donor tissue to be successful. The Missouri Osteochondral Allograft Preservation System (MOPS) may extend the time when stored osteochondral tissues remain viable. This study aimed to provide an independent evaluation of MOPS storage by evaluating chondrocyte viability, chondrocyte metabolism, and the cartilage extracellular matrix using an ovine model.

Femoral condyles from twelve female Arcott sheep (6 years, 70 ± 15 kg) were assigned to storage times of 0 (control), 14, 28, or 56 days. Sheep were assigned to standard of care [SOC, Lactated Ringer's solution, cefazolin (1 g/L), bacitracin (50,000 U/L), 4°C storage] or MOPS [proprietary media, 22-25°C storage]. Samples underwent weekly media changes. Chondrocyte viability was assessed using Calcein AM/Ethidium Homodimer and reported as percent live cells and viable cell density (VCD). Metabolism was evaluated with the Alamar blue assay and reported as Relative Fluorescent Units (RFU)/mg. Electromechanical properties were measured with the Arthro-BST, a device used to non-destructively compress cartilage and calculate a quantitative parameter (QP) that is inversely proportional to stiffness. Proteoglycan content was quantified using the dimethylmethylene blue assay of digested cartilage and distribution visualized by Safranin-O/Fast Green staining of histological sections. A two-way ANOVA and Tukey's post hoc were performed.

Compared to controls, MOPS samples had fewer live cells (p=0.0002) and lower VCD (p=0.0004) after 56 days of storage, while SOC samples had fewer live cells (p=0.0004, 28 days; p=0.0002, 56 days) and lower VCD (p=0.0002, 28 days; p=0.0001, 56 days) after both 28 and 56 days (Table 1). At 14 days, the percentage of viable cells in SOC samples were statistically the same as controls but VCD was lower (p=0.0197). Cell metabolism in MOPS samples remained the same over the study duration but SOC had lower RFU/mg after 28 (p=0.0005) and 56 (p=0.0001) days in storage compared to controls. These data show that MOPS maintained viability up to 28 days yet metabolism was sustained for 56 days, suggesting that the conditions provided by MOPS storage allowed fewer cells to achieve the same metabolic levels as fresh cartilage. Electromechanical QP measurements revealed no differences between storage methods at any individual time point. QP data could not be used to interpret changes over time because a mix of medial and lateral condyles were used and they have intrinsically different properties. Proteoglycan content in MOPS samples remained the same over time but SOC was significantly lower after 56 days (p=0.0086) compared to controls. Safranin-O/Fast Green showed proteoglycan diminished gradually beginning at the articular surface and progressing towards bone in SOC samples, while MOPS maintained proteoglycan over the study duration (Figure 1).

MOPS exhibited superior viability, metabolic activity and proteoglycan retention compared to SOC, but did not maintain viability for 56 days. Elucidating the effects of prolonged MOPS storage on cartilage properties supports efforts to increase the supply of fresh osteochondral allografts for clinical use.

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Surgical reattachment of torn rotator cuff tendons can lead to satisfactory clinical outcome but failures remain common. Ortho-R product is a freeze-dried formulation of chitosan (CS) that is solubilized in platelet-rich plasma (PRP) to form injectable implants. The purpose of the current pilot study was to determine Ortho-R implant acute residency, test safety of different implant doses, and assess efficacy over standard of care in a sheep model.

The infraspinatus tendon (ISP) was detached and immediately repaired in 22 skeletally mature ewes. Repair was done with four suture anchors in a suture bridge configuration (n = 6 controls). Freeze-dried formulations containing 1% w/v chitosan (number average molar mass 35 kDa and degree of deacetylation 83%) with 1% w/v trehalose (as lyoprotectant) and 42.2 mM calcium chloride (as clot activator) were solubilized with autologous leukocyte-rich PRP and injected at the tendon-bone interface and on top of the repaired site (n = 6 with a 1 mL dose and n = 6 with a 2 mL dose). Acute implant residency was assessed histologically at 1 day (n = 2 with a 1 mL dose and n = 2 with a 2 mL dose). Outcome measures included MRI assessment at baseline, 6 weeks and 12 weeks, histopathology at 12 weeks and clinical pathology. MRI images and histological slides were scored by 2 blinded readers (veterinarian and human radiologist, and veterinarian pathologist) and averaged. The Generalized Linear Model task (SAS Enterprise Guide 7.1 and SAS 9.4) was used to compare the different groups with post-hoc analysis to test for pairwise differences.

Ortho-R implants were detected near the enthesis, near the top of the anchors holes and at the surface of ISP tendon and muscle at 1 day. Numerous polymorphonuclear cells were recruited to the implant in the case of ISP tendon and muscle. On MRI, all repair sites were hyperintense compared to normal tendon at 6 weeks and only 1 out 18 repair sites was isointense at 12 weeks. The tendon repair site gap seen on MRI, which is the length of the hyperintense region between the greater tuberosity and tendon with normal signal intensity, was decreased by treatment with the 2 mL dose when compared to control at 12 weeks (p = 0.01). Histologically, none of the repair sites were structurally normal. A trend of improved structural organization of the tendon (p = 0.06) and improved structural appearance of the enthesis (p = 0.1) with 2 mL dose treatment compared to control was seen at 12 weeks. There was no treatment-specific effect on all standard safety outcome measures, which suggests high safety.

Ortho-R implants (2 mL dose) modulated the rotator cuff healing processes in this large animal model. The promising MRI and histological findings may translate into improved mechanical performance, which will be assessed in a future study with a larger number of animals. This study provides preliminary evidence on the safety and efficacy of Ortho-R implants in a large animal model that could potentially be translated to a clinical setting.

Osteoarthritis (OA) is a debilitating disease characterised by degradation of articular cartilage and subchondral bone remodeling. Current therapies for early or midstage disease do not regenerate articular cartilage, or fail to integrate the repair tissue with host tissue, and therefore there is great interest in developing biological approaches to cartilage repair. We have shown previously that platelet-rich plasma (PRP) can enhance cartilage tissue formation. PRP is obtained from a patient's own blood, and is an autologous source of many growth factors and other molecules which may aid in healing. This raised the question as to whether PRP could enhance cartilage integration. We hypothesise that PRP will enhance integration of bioengineered cartilage with native cartilage.

Chondrocytes were isolated from bovine metacarpal-phalangeal joints, seeded on a porous bone substitute (calcium polyphosphate) and grown in the presence of FBS to form an in vitro model of osteochondral-like tissue. After 7 days, the biphasic constructs were soaked in PRP for 30 minutes prior to implantation into the core of a ring-shaped biphasic explant of native bovine cartilage and bone. Controls were not soaked in PRP. The resulting implant-explant construct was cultured in a stirring bioreactor in serum free conditions for 2 weeks. The integration zone was visualised histologically. A push-out test was performed to assess the strength of integration. Matrix accumulation at the zone of integration was assessed biochemically and the gene expression of the cells in this region was assessed by RT-PCR. Significance (p<0.05) was assessed by a student's t-test or one-way ANOVA with tukey's post hoc.

PRP soaked bioengineered implants, integrated with the host tissue in 73% of samples, whereas control bioengineered implants only integrated in 19% of samples based on macroscopic evaluation (p<0.05). The integration strength, as determined by the normalised maximum force to failure, was significantly increased in the PRP soaked implant group compared to controls (219 +/− 35.4 kPa and 72.0 +/− 28.5 kPa, respectively, p<0.05). This correlated with an increase in glycosaminoglycan and collagen accumulation in the region of integration in the PRP treated implant group, compared to untreated controls after 2 weeks (p<0.05). Immunohistochemical studies revealed that the integration zone was rich in collagen type II and aggrecan. The cells at the zone of integration in the PRP soaked group had a 2.5 fold increase in aggrecan gene expression (p=0.05) and a 3.5 fold increase in matrix metalloproteinase 13 expression (p<0.05) compared to controls.

PRP soaked bio-engineered cartilage implants showed improved integration with native cartilage compared to non-treated implants, perhaps due to the increased matrix accumulation and remodeling at the interface. Further evaluation is required to determine if PRP improves integration in vivo.

Purpose:

To develop a method for depth-wise analysis of subchondral bone that considers the gradient of bone volume, density and organization between the articular surface and the marrow cavity.

Method: Since 30% of patients fail microfracture for contained chondral lesions, our hypothesis was that early subchondral sclerosis increases compaction of bone around microfracture holes, leading to failed cartilage repair. Human osteochondral segments from the knee joint were characterized macroscopically using the Outerbridge score, then imaged at 45 micron resolution using microCT. Regions of interest (ROI) were chosen under normal cartilage and abnormal cartilage (Outerbridge Score=1). Routine Bone mineral density (BMD) analysis was performed on each ROI using GE MicroView™ analysis software. Additional depth-wise analysis of BMD was done by exporting each ROI was a density map, and calculating the mean, standard deviation and rate of change of BMD by slice in the vertical (coronal) plane. Plots of normal and early OA data by depth were compared. Microfracture holes were made in normal and sclerotic subchondral bone, and depth-wise measurements of subchondral compaction around the holes were made were made.

Results: Bone under normal versus OA cartilage was very subtly different in microCT images, but ROI microCT analysis showed that the OA samples were more mineralized and contained more bone. Using the depth-wise analysis algorithm, automated detection and measurement of the subchondral bone plate and other discrete structures was possible. The depth-wise analysis confirmed that the osteoarthritic subchondral bone plate had a higher BMD and bone volume fraction, but also showed that the rate of change (gradient) in BMD was greater. Horizontally orientated trabeculae and other anomalies were found in OA bone that contributed a more variable BMD in trabecular bone at up to 5 mm from the articular surface. Bone with early sclerotic changes had significantly (p< .01) more bone volume fraction and BMD (p< .05) around microfracture holes in this ex vivo experiment.

Conclusion: An enhanced picture of subchondral bone plate and trabecular bone anomalies can be appreciated using a depth-wise approach to image analysis. Both sclerosis and osteopenia have been reported in OA and models of OA, but this analysis shows that variability and gradient of BMD change adjacent to the articular cartilage is a significant feature of OA. This is consistent with some theories of OA progression that implicate stress concentration between the cartilage and subchondral bone plate leading to cartilage degeneration. More importantly, bone sclerosis has a direct effect on the amount of compaction around microfracture holes, so improvements in microfracture technique are needed to avoid this.

Purpose: The mechanical function and strain behavior of the knee meniscus is not fully understood, due to multiple tissues with disparate properties, as well as complex contact patterns and intricate loading mechanisms. More comprehensive understanding of joint mechanics may contribute to improved treatment options for patients with injuries and osteoarthritis. There is very limited information available on the 3D strain of the intact meniscus. The objective of this work was to use mCT with copper microsphere markers to quantify three-dimensional strain of the meniscus under physiologic loading.

Method: Two healthy fresh frozen ovine knee specimens were harvested. Copper microspheres (0.5mm) were injected into anterior and posterior tetrahedral clusters in the medial meniscus using 20-gauge hypodermic needles. Needle cavities were sealed with ovine tendon tissue. Joints were loaded to 100% body weight in a 4 DOF CT-compatible pneumatically-driven device with flexion angles ranging from 62–98°. Images were acquired with an eXplore Locus Ultra mCT scanner and reconstructed with commercial software. A time series of images were acquired with the joint unloaded, during static loading, and at a reduced load (25% BW).

Results: The average maximum principle strains in the anterior element of the two specimens at 62o of flexion increased by 21% during loading and decreased by 13% during unloading. The maximum principle strains were 28% larger in the anterior element than the posterior. The strains in the anterior element decreased by 6.5% with time following load application, and decreased by 16% with load reduction, yielding relatively low residual strain. Strains were 2% larger in the anterior portion with larger flexion angles.

Conclusion: The objective of this work was to develop a reliable method for quantifying 3D strains in the meniscus. Results support the notion that mCT imaging with copper microspheres in the meniscus may be a viable technique for more comprehensive 3D strain analysis. The relatively low residual strains measured in this study indicate that copper microspheres are stable markers in this application. This technique may be useful in directing future studies aimed at understanding the impact of meniscal pathologies and the success of repair techniques.

Purpose: To determine if an adenovirus vector expressing BMP-7 can alter the progression of post-traumatic osteoarthritis.

Method: Preliminary dose-response studies were done in ovine metacarpal-phalangeal joints using 10^9, 10^10, and 10^11 virus particles (VP). In-vitro transfection efficiency studies were done using ovine synovial cells, chondrocytes and HEK293 cells. In-vivo studies were conducted in 16 sheep that underwent surgery to create bilateral contusive impact injuries to the medial femoral condyle. One week later 10^9 VP were injected into one joint of each sheep, while four sheep remained untreated bilateral controls. Three months later the sheep were sacrificed for assessments including histological scoring, cartilage glycosaminoglycan assays, and immunostaining for Col2 3/4 short collagen fragments that are generated by metalloproteinases during OA progression.

Results: Transfection with 10^9 VP produced slightly longer expression than higher concentrations of VP. HEK293 cells expressed BMP-7 quickly but synoviocytes and chondrocytes expressed this protein at 48 and 96 hours. Knee joints that received Ad5-BMP-7 produced up to 2.5 ng of BMP-7 between day seven and 21. These joints had reduced cartilage degneration at the injury sites and less centrifugal progression of OA across the femoral condyle. Histological scores were reduced as was Col2 C3/4 short immunostaining.

Conclusion: BMP-7 has a homeostatic role in cartilage and can be used therapeutically¹. Ad5-BMP-7 transfection of synovial tissue produced sufficient BMP-7 to stop the progression of degenerative changes after trauma that would usually lead to OA. Adenoviral vectors can create inflammation and neutralizing antibodies but these complications were minimized by using a low (10^9) dose. Human trials using similar vectors are ongoing and the outcome of these will determine whether gene therapy will become a useful tool when patients are at risk of post-traumatic OA.

Two fixation devices for rotator cuff repair were compared in a sheep model. Surgical transection of the supra-spinatus tendon insertion was repaired using metallic OBL suture anchors or Suretac II anchors. Twelve weeks postoperatively the repair site was assessed using histology, polarized light microscopy and biomechanical testing. No important differences were found between these two repair methods.

The purpose of this study was to compare traditional rotator cuff fixation devices with bioabsorbable press-fit tacks.

Sixteen sheep were assigned to OBL (n=8) or Suretac (n=8) treatment groups. Four sheep shoulder joints were used as unoperated controls. Treated sheep underwent general anesthesia and a lateral arthrotomy using aseptic technique to allow transection of the supraspinatus tendon insertion. The tendon-bone interface was repaired with two fixation devices according to the manufacturer’s directions. After recovery from anesthesia the sheep were maintained in small pens for twelve weeks. After sacrifice, muscle-tendon-bone blocks were prepared for mechanical testing. The specimen underwent a preload of 25N, followed by cyclic loading (10–50N x10), then loading at 480mm/min until failure. The remaining bone-tendon interface was fixed, embedded in plastic and 100μ undecalcified histological sections were cut, polished and stained.

All tendons had healed to the humerus and the repair site was two to three times larger than unoperated controls. There were no significant differences between the two treatment groups with respect to maximum load, modulus, and energy per unit area. Histological analysis is ongoing.

These data suggest that these two fixation methods are functionally equivalent in this model. Press-fit fixation devices do not knot tying and they can be inserted arthroscopically so they are a convenient fixation method.

This study confirms that press fit anchors and metallic anchors with sutures are equivalent for repair of bone-tendon interfaces.

Funding: Smith & Nephew supplied the fixation devices for this project. Thanks to Deb McWade for technical assistance.

Osteochondral autogenous transfer is an accepted treatment for the management of osteochondral defects in the knee. Concerns about donor site morbidity and kissing lesions of the patella lead us to assess the efficacy of filling donor sites with bioabsorbable bone cement in a sheep model. Donor sites were assessed two, eight and sixteen weeks postoperatively using macroscopic scoring, histology and creep indentation testing. At eight and sixteen weeks after graft harvest there were fewer patellar kissing lesions in the treatment group. Control defects had more extrinsic repair whereas cartilage flow was the predominate source of repair tissue in the treated group.

The purpose of this study was to determine if Norian SRS^® bone cement can mitigate donor site morbidity in a model of osteochondral transplantation in the knee.

Ten sheep were assigned to either a control or experimental group. Under general anesthesia and aseptic technique, four donor site defects were created in standardized non-weightbearing regions of the trochlear ridge. These defects measured 4.5 mm in diameter x 10 mm deep. Norian SRS bone cement was used to fill donor site defects up to the level of the tidemark in five experimental sheep. In the control group (n=5), donor sites were left unfilled. One sheep from each group was sacrificed two weeks postoperatively and two sheep from each group were sacrificed at eight and sixteen weeks. Macroscopic scoring, histology and biomechanical creep indentation were used to assess the knee joints.

At eight weeks, treated defects had more filling by facilitating cartilage flow, yielding fewer kissing lesions on the patella. At sixteen weeks, the treated group had more cartilage flow but little extrinsic repair. The control group defects had a more uniform fill with repair tissue and better biomechanical properties but kissing lesions on the patella remained problem.

Short-term results suggest that unfilled donor sites allow better extrinsic repair at the expense of creating kissing lesions. Norian SRS cement reduced kissing lesions on the patella.

Norian SRS was a barrier to extrinsic repair but supported cartilage flow. Cartilage flow in thicker, human cartilage will be greater. It may be possible to support cartilage flow and still allow extrinsic repair by applying Norian SRS up to but not including the tidemark.

Funding: The authors received funding from Synthes, Canada for this project. Thanks to Deb McWade for technical assistance.