Orthopaedic Research Laboratory Alumni Council

Alumni Laboratories

Visit the research laboratories of the alumni of Dr. Woo.

Board of Directors

Richard Debski, Ph.D. President
Caroline Wang, M.S. Secretary
Jamie Pfaeffle, M.D., Ph.D. Treasurer
Doug Boardman, M.D.
Thay Lee, Ph.D.
Patrick McMahon, M.D.
Karen Ohland, M.S.
Christos Papgeorgiou, M.D.
Masataka Sakane, M.D.
Sven Scheffler, M.D.
Jennifer Wayne, Ph.D.

2004 Mrs. Ho-Tung Cheong Grant Recipient

Sarita Maheedhara
University of Pittsburgh

Determination of Biomechanical and Histological Properties of SIS Repaired Patellar Tendon


Sarita with her advisor, Dr. Woo

Acknowledgement

I was grateful and honored to receive Mrs. Ho-Tung Cheong Grant from ORLAC for 2004. The generous support enabled me to initiate an animal study to investigate the effect of porcine small intestine submucosa (SIS) on the histological appearance, tissue dimensions, and structural properties of the healing patellar tendon (PT) tissue up to 12 weeks after creation of a central third defect in a rabbit model. This multidisciplinary project was successfully completed by a research group that involved clinicians as well as graduate students, under the supervision of Dr. Savio Woo and Dr. Steve Abramowitch. I would like to thank the above-mentioned people for their help and advice throughout this project and the whole MSRC family for their support during my research experience at this laboratory.

Progress Report

The motivation for this project was to improve healing to alleviate the post-operative complications associated with ACL reconstruction with middle third of patellar tendon. It is well known that ACL does not heal by itself and requires surgery; and is often reconstructed with the middle third of the patellar tendon as an autograft. The remaining patellar tendon heals with an inferior morphological appearance and mechanical properties, thereby resulting in weak extensor mechanism and patellofemoral problems. Currently, the patellar tendon defect is either left open or is sutured, and it has been reported that there is no significant difference between the treatment modalities. Both result in shortening of the tendon, which leads to several other complications, such as anterior knee pain and osteoarthritis. Recently, it has been demonstrated at MSRC that a bioscaffold, namely porcine small intestine submucosa (SIS) improved healing of an isolated MCL gap injury in a rabbit model. Since SIS is a biologically active tissue scaffold composed primarily of type I collagen, we hypothesized that SIS treatment improves the histology as well as biomechanical properties of the healing patellar tendon at 12 weeks. The animal model had to be switched from goat to rabbit in order to meet our objectives within the provided budget.

This study aims at collecting preliminary data that would support further funded investigation. 24 skeletally mature rabbits were used for this study, where the central third defect (3mm) was created in the right PT, while left knees served as a sham controls.

In the SIS-treated group (n=12), two strips of SIS were sutured to the anterior and posterior sides of the PT defect. The defect was left open in the non-treated group. Three samples for each group at 3 and 12 weeks (12 in total) were used for histology. Six samples for each treatment group (12 in total) were used for CSA measurement and biomechanical testing at 12 weeks.

Application of the functional tissue engineered SIS scaffold to the damaged tissue will enhance our understanding of the mechanisms of tendon healing; thus, help to develop a better clinical management strategy with improved outcomes. Ultimately, we believe the knowledge gained will help to develop new ways of treating patellar tendon defects in order to maintain their function.

For histology, 6 µm thick serial frozen sections were cut within the sutures to ensure the sections consisted of healing tissue. Slides were stained with Masson's trichrome to visualize collagen content and distribution. For the specimens utilized for biomechanical testing, the healing tissue was isolated via sharp dissection using the sutures to define the width. A standard width of 2 mm was used to dissect sham tissues. CSA was measured using a laser micrometer. Reflective markers were placed on the PT midsubstance for strain measurements, which were recorded using a Motion Analysis(TM) video system. The healing central BPTB complexes were fixed to an Instron(TM) in custommade clamps, preloaded to 2N, preconditioned for 10 cycles from 0 to 1 mm, and loaded to failure. An elongation rate of 10 mm/min was used for all procedures. Structural properties of the healing central BPTB complex (stiffness and ultimate load) and mechanical properties of the healing PT tissue (tangent modulus and ultimate tensile strength) were obtained from the resulting load-elongation and stress-strain curves, respectively. The maximum slope over a 1 mm interval of elongation and a 2% interval of strain were utilized to determine stiffness and tangent modulus, respectively. An unpaired t-test was used to compare treatment groups. A paired t-test was utilized to compare treatment groups to their respective sham controls. A Bonferroni adjustment was utilized to correct for multiple comparisons. Thus, significance was set at p<0.03.

Gross observations revealed that all defects from both treatment groups filled with new tissue and showed edema at 3 weeks. The NT tissues displayed concave defects, while the SIS-treated tissues possessed defects with no concavity and a larger width at 12 weeks. Histological examination at 3 weeks showed a loose, disorganized fibrous tissue with low cellularity in the NT group. In contrast, defects of SIS-treated samples showed areas of high cellularity (Fig. 1A). At 12 weeks in the NT group, a sparse distribution of cells was observed, and collagen staining was seen in patches. Conversely, the SIS group appeared to contain a large number of spindle shaped cells within an organized collagen matrix (Fig. 1B).

CSA measurements of the healing tissue measured 56% greater in the SIS-treated group compared to those in the NT group (5.0 ± 2.2 mm2 vs. 3.2 ± 1.3 mm2, respectively), but this result was not significant (p=0.13). During load to failure tests, all specimens failed in the midsubstance, except for one sham specimen for each group, which failed at the patellar insertion. SIS-treatment showed trends of increasing stiffness (p=0.27) and ultimate load (p=0.15) of the healing central BPTB complex by 40% and 55%, respectively ( Table 1 ). Tangent modulus and ultimate tensile strength were similar between healing PT tissues of the SIS-treated and NT groups (p>0.03, Table 1 ). All parameters representing the structural and mechanical properties for each treatment group were significantly lower compared to their respective sham controls (p<0.03, Table 1 ).

Based on the results of this study, SIS-treatment shows the potential to increase the quantity of healing tissue and structural properties of the healing central BPTB complex after a surgically created central third PT defect, supporting our hypothesis. Most importantly, the observation of earlier tissue growth and aligned collagen fibers at 12 weeks in the SIS-treated group suggests that SIS has positively changed the healing response of the PT. Therefore, further investigation is warranted for this application. To this end, longer-term studies focusing on PT healing with SIS-treatment are underway as well as experiments aimed at enhancing the healing potential of the scaffold prior to implantation via cell seeding and mechanical conditioning. Ultimately, the goal is to improve both the quantity and quality of the healing tissue within the PT defect in an effort to reduce donor site morbidity following BPTB graft harvest.

Structural Properties of the Healing Central BPTB Complex

Sham

SIS-treated

NT

Stiffness (N/mm)

151.6 ± 21.9 a

33.9 ± 14.0

24.3 ± 14.9

Ultimate Load (N)

250.3 ± 69.0 a

67.7 ± 25.8

43.8 ± 27.4

Mechanical Properties of the Healing PT Tissue

Sham

SIS-treated

NT

Tangent Modulus (Mpa)

1435.6 ± 568.8 a

238.1 ± 88.0

213.4 ± 99.7

Ultimate Tensile Strength (Mpa)

71.9 ± 11.2 a

14.0 ± 4.5

14.1 ± 9.0

Table 1. Parameters representing biomechanical properties for sham (n=12), SIS-treated (n=6), and NT (n=6) groups at 12 weeks (mean ± std). a= significantly different from treated groups (p<0.03).

© Orthopaedic Research Laboratory Alumni Council 2013. | Template Design by Download Website Templates