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The response of rabbit patellar tendons after autologous blood injection

TAYLOR, MARK A.; NORMAN, TIMOTHY L.; CLOVIS, NINA B.; DAVID BLAHA, J.

Medicine & Science in Sports & Exercise: January 2002 - Volume 34 - Issue 1 - p 70-73
BASIC SCIENCES: Original Investigations

TAYLOR, M. A., T. L. NORMAN, N. B. CLOVIS, and J. D. BLAHA. The response of rabbit patellar tendons after autologous blood injection. Med. Sci. Sports Exerc., Vol. 34, No. 1, 2002, pp. 70–73.

Purpose Blood is a rich source of growth factors that can stimulate fibrocyte migration and help induce neovascular ingrowth. These properties may be able to stimulate a healing response in chronic degeneration of a tendon (tendonosis). The purpose of this study was to assess the biomechanical and histological effects of autologous blood injection on animal tendons.

Methods New Zealand white rabbit left side patellar tendons were injected with 0.15 cc of autologous blood. We then compared the mechanical properties and histology to the normal right patellar tendon at 6 and 12 wk.

Results At 6 and 12 wk after the injection, there were no differences in the histology compared with normal tendon tissue, and there were no significant changes in tendon stiffness. Biomechanically, the tendons were not damaged at 6 wk after the injection. By 12 wk, tendons that were injected with blood were significantly (P < 0.014) stronger.

Conclusion We found that injecting blood directly into normal tendons appears safe. Further evaluation of this technique would appear indicated.

Musculoskeletal Research Center, Department of Orthopedics and Department of Mechanical and Aerospace Engineering, West Virginia University, Morgantown, WV 26506-9196

Submitted for publication December 2000.

Accepted for publication March 2001.

Chronic tendon disorders are a common source of disability from recreational and vocational activities (16,18). Although most respond to conservative care, some chronic tendon disorders do not heal well and are a source of chronic symptoms. Even prolonged periods of activity modification may not allow adequate healing for normal activity resumption (16,18). Chronic tendon disorders are more common in middle-aged adults (1,17,23). Decreased blood supply to the tendon is felt to play a role in the development of tendon degeneration (tendonosis) (1,10,13,14,22).

Those patients who do not respond to conservative care have limited options. Injection of tendons with steroids is felt to increase the risk of tendon rupture (5,11). Laser phototherapy (25) and stem cell therapy (30) are being evaluated but are not in widespread clinical use. Various forms of surgical intervention have met with some success (6,17,20,23). Blazina et al. (6) noted that patients who had symptomatic tendon disorders and went on to develop a rupture of that tendon often had resolution of their previous symptoms. Bleeding may stimulate new vascular ingrowth and fibroblast activity (17).

Despite the disabling conditions associated with tendon disorders, there are no known reliable animal models that we felt were appropriate to study this procedure. To prove a treatment is effective, a long-term histological and biomechanical model demonstrating a chronic nonhealing tendon lesion persisting beyond an experimental treatment is needed (15,4,19). We have selected the rabbit patellar tendon complex in older male animals as our model to determine the safety of the technique before any further study. The objective of this study is to evaluate the long-term biomechanical and histologic effects injected blood has on normal tendon tissue.

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MATERIALS AND METHODS

The policies and procedures in the Guide for the Care and Use of Laboratory Animals are the policy of our institution. The animals were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care (AAALAC). After protocol approval by the institutional Animal Care and Use Committee, postbreeding male New Zealand white rabbits were used for the study. Animals were anesthetized before injections with intramuscular xylazine (5 mg·kg−1) and ketamine (44 mg·kg−1). At the end of the study periods, the animals were euthanized with xylazine (0.5 cc) and ketamine (1.0 cc) intramuscular and 2.5 cc of sodium pentobarbital in 10% isopropyl alcohol intracardiac. Seven untreated rabbits were used as a baseline control group. Twenty-eight rabbits were injected with 0.15 cc of autologous blood into the left patellar tendon. The right patellar tendon was used as a matched control. All injections were performed by the lead author using a 25-gauge needle. Fourteen treated rabbits were sacrificed at 6 wk post injection. The final 14 were sacrificed at 12 wk post injection. At 6 and 12 wk, 11 rabbits were used for mechanical testing and three for histology.

Mechanical testing was performed on a Materials testing System (MTS Systems, Minneapolis, MN) at a rate of 10 mm·min−1 until failure (29). Immediately after sacrifice, each patella-patellar tendon-proximal tibia complex was dissected and tested. The patella was stabilized using two pins arranged perpendicular to the patella, and the tibial plateau was held in place by a fixture mounted to the testing machine. The tendon was then loaded in tension until failure. Load and displacement were recorded. Stiffness was calculated as the slope of the linear portion of load displacement.

Light microscopy analysis of the tendons was performed for qualitative histological assessment. The tendons were fixed for 24 h in 10% neutral buffered formalin. After fixation, the specimens were placed in 12% formic acid for approximately 1 wk for decalcification and then washed and placed in an automated tissue processor for processing. After paraffin embedding, the specimens were sectioned at 5 microns and then stained with routine H&E staining techniques. Once prepared, the histologic specimens were reviewed by an animal pathologist.

Statistical evaluation for mechanical strength changes were performed using two-tailed, paired Student t-tests by using the statistical package JMP (SAS Institute Inc., Cary, NC). Results are reported as mean ± standard deviation.

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RESULTS

None of the rabbits showed any ill effect from the injections. Upon awakening from anesthesia, they resumed normal activity immediately. There were no infections or other reactions related to the injections. With the exception of one 12-wk specimen, all of the tendons ruptured at the inferior pole of the patella. The exception in the 12-wk group ruptured both tendons at the tibial tubercle. One of the 6-wk specimens sustained a tibia fracture during preparation, and data were not collected for this animal.

Results of the mechanical tests on the patella-patellar tendon-tibia complexes are summarized in Table 1. The differences in strength of the tendon complexes (blood injected minus uninjected) are also illustrated (Fig. 1). In the baseline control group, there was a nonsignificant [P = 0.74, CI: (−99.6, 132.9)] difference between left (658 ± 152) and right (641 ± 49) tendon complexes. At 6 wk after the injection of autologous blood into the left tendon (734 ± 115 N), there was a nonsignificant [P = 0.406, CI: (−52.5, 118.3)] increase in strength compared with the uninjected control tendon (701 ± 146 N). However, at 12 wk after the injection of blood, the injected left patellar tendon complex (741 ± 98 N) was significantly [P < 0.014, CI: (24.8, 174.5)] stronger than the uninjected tendon complexes (642 ± 106 N). Although not significant, the statistical analysis suggested an early trend toward increasing stiffness at 6 wk: the injected left (193 ± 21) was stiffer [P < 0.054, CI: (−0.31, 31.5)] than the control right (177 ± 23). However, at 12 wk this trend did not progress. There was no significant [P < 0.547, CI: (−56.4, 33.0)] difference between the stiffness of baseline left (167 ± 28) and baseline right (178 ± 38) tendon complexes.

Table 1

Table 1

FIGURE 1

FIGURE 1

Upon microscopic evaluation at low and high magnification, no abnormality or structural change could be identified in the arrangement of the collagen in the patellar tendon complexes (Fig. 2). There was no evidence of vascular changes or inflammatory cells. The two sides appeared identical histologically.

FIGURE 2

FIGURE 2

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DISCUSSION

Several different surgical techniques have been described for chronic tendon problems. Debridement of damaged tissue is sometimes the primary goal (17,24). One technique, longitudinal tenotomy, has a primary goal of creating bleeding within the tendon (17,20). Blood is a rich source of growth factors, which can stimulate fibrocyte migration and activation (8,9). Additionally, there are factors in blood, such as endothelial cell growth factor, that can help induce neovascular ingrowth (12,21). Clotted blood has been used both experimentally and clinically to stimulate fibrous tissue healing in the meniscus model (2,3,26,28). We found one published article using blood products (fibrin glue) to augment the repair of Achilles tendon ruptures. In a poster exhibit, Hansen et al. (7) reported using autologous blood injection on 22 patients with lateral epicondylitis. They had all failed conservative care. Their success rate was 86% with no apparent complications. Other forms of injection therapy are being used clinically. There is anecdotal evidence that injecting dextrose-phenol solution (prolotherapy) may be of help in treating chronic tendon problems in addition to other areas of chronic pain (27).

The purpose of this study was to identify long-term safety of blood injection. As such, we did not look at the early inflammatory stages of soft tissue healing, including tenocyte activation and collagen deposition. By 6 wk, the soft tissue healing process would have been in the maturation and remodeling stage. This would explain the normal histology. This maturation of the collagen would have continued between 6 and 12 wk (17). We hypothesize this collagen maturation explains the increase in strength between 6 and 12 wk.

The objective of this study was to assess the safety and biomechanical and histological effects of autologous blood injection on animal tendons. We found that injecting blood directly into normal tendons caused no apparent harm. At 6 and 12 wk after the injection, there was no difference in the histology from normal tendon tissue. Biomechanically, at 6 wk there was an early trend for increased stiffness and strength; however, the tendons were statistically stronger (15.5%) at 12 wk after blood injection compared with controls without further increases stiffness. Our data suggest that further evaluation of injecting blood into a chronically nonhealing tendon would be safe.

This project was funded by a grant from Mark Taylor, M.D. We are grateful to Ms. Suzanne Smith for her assistance with manuscript preparation, Mr. Vince Kish for performing the mechanical tests, Gerry Hobbs, Ph.D., Department of Statistics, for statistical support, and Patsy Willard and Dr. Choudari Kommineni, NIOSH of Morgantown, WV, for assistance with the histological preparation and analysis.

Address for correspondence: Timothy L. Norman, Ph.D., Department of Orthopedics, P.O. Box 9196 Health Sciences Center, West Virginia University, Morgantown, WV 26506-9196; E-mail: tnorman@wvu.edu.

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Keywords:

TENDON DEGENERATION; TENDONOSIS; HEALING; AUTOLOGOUS BLOOD INJECTION

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