Nembutal

The healing of a hamstring graft to bone is the weak link in the reconstruction of a cruciate ligament using this donor material. We therefore investigated the augmentation of healing at the tendon-bone interface using calcium-phosphate cement (CPC).

We performed semitendinosus autograft reconstructions of the anterior cruciate ligament on both knees of 22 New Zealand white rabbits. The interface between the grafted tendon and the bone tunnel for one knee was filled with CPC. Six rabbits were killed at the end of the first and second post-operative weeks in order to evaluate the biomechanical changes. Two rabbits were then killed sequentially at the end of weeks 1, 3, 6, 12 and 24 after operation and tissue removed for serial histological observation.

Histological examination showed that the use of CPC produced early, diffuse and massive bone ingrowth. By contrast, in the non-CPC group of rabbits only a thin layer of new bone was seen. Mechanical pull-out testing at one week showed that the mean maximal tensile strength was 6.505 ± 1.333 N for the CPC group and 2.048 ± 0.950 N for the non-CPC group. At two weeks the values were 11.491 ± 2.865 N and 5.452 ± 3.955 N, respectively.

Our findings indicate that CPC is a potentially promising material in clinical practice as regards its ability to reinforce the fixation of the tendon attachment to bone and to augment the overall effectiveness of tendon healing to bone.

In many surgical reconstructions an autograft tendon is passed through a bone tunnel. Previous studies on biomechanical pullout have shown that immediately after reconstruction most failures have occurred at the anchoring site of the tendon despite the various methods used.1,2 Therefore, the effectiveness of the healing between bone and tendon greatly influences the efficacy of post-operative rehabilitation programmes and ultimately the clinical results.

While healing of both bone and tendon has been well studied in the past, few studies have investigated the specific healing of grafted tendon to bone. In 1938 and 1942, Kernwein, Fahey and Garrison3 and Kernwein4 reported in a rabbit model that grafted tendon was attached to bone by means of ossification and the incorporation of fragments of the tendon into the bone material. In 1993, Rodeo et al5 found that the progressive increase in strength of the bone-tendon interface subsequent to repair appeared to correlate with the degree of bone ingrowth, mineralisation and the level of maturation of the tissue. In 1988, Arnoczky et al6 noted that the maximum values for pullout strength were proportional to the degree of osseous ingrowth into the interfacial gap. From these studies, it appeared that the level of hone ingrowth was the most important factor which influenced the mechanical strength of interfacial healing. We hypothesised that filling of the gap between the grafted tendon and the bone tunnel with an osteoconductive material would augment the healing by inducing more bone ingrowth at this site. Our aim therefore was to evaluate in a rabbit model whether filling the bone-tendon gap with calcium-phosphate cement (CPC) would encourage healing.

Materials and Methods

We used 22 adult male New Zealand White rabbits with an approximate weight of 4.0 kg. They were kept in cages measuring 90 cm × 45 cm × 45 cm and activity was allowed only in the cages.

The semitendinosus tendon was chosen for reconstruction of the anterior cruciate ligament (ACL) because of its uniformity of size and length.1 After the rabbits had been anaesthetised with intravenous Nembutal (45 mg/ kg), an anteromedial incision was made through the medial retinaculum. The ACL was excised and a bone tunnel, with a diameter of 2.4 mm, was made in the proximal tibia just anterior to the medial collateral ligament (MCL) ending at the original point of insertion of the ACL on the intercondylar tibial spine. A femoral tunnel of the same size was made from the midline of the intercondylar notch ending just superior to the origin of the lateral collateral ligament (LCL). The semitendinosus tendon was dissected and passed through the tibial and femoral bone tunnels and sutured to the LCL. The procedure was repeated for the other knee. The interface between the grafted tendon and the femoral bone tunnel of one knee was filled with 0.5 ml of CPC injected into the interface from a 2 ml syringe with a 19-gauge needle through the extrusion orifice of the femoral bone tunnel.

The limbs were not immobilised after completion of the surgical procedures and the rabbits were allowed to undertake normal activity within the cage. The results of a similar study1 indicated that after three weeks the intra-articular portion of the graft ruptured when tested mechanically. In order to ensure that the failure point would occur at the interface between the grafted tendon and bone, six rabbits were killed at the end of the first and the second weeks. For serial histological observation of the interface two rabbits each were then killed sequentially at the end of weeks 1, 3, 6, 12 and 24.

Calcium-phosphate cement (CPC). The CPC used in our study was obtained from mixing equimolar Ca4 (PO4^sub ^)2^sub ^O (TTCP) and dried CaHPO^sub 4^ (DCPA) powder in a 1M phosphate-containing solution. The Ca^sub 4^ (PO^sub 4^)^sub 2^O (TTCP) powder used for the preparation of the non-dispersive CPC (nd-CPC) for our study was made according to the method of Brown and Epstein.7 The dried CaHPO^sub 4^ (DCPA) powder used for the preparation of the CPC material was obtained as a commercial product (Acros Organics, Fairlawn, New Jersey). In order to obtain a cement paste which could be easily handled, the TTCP/DCPA particles were mixed in a IM phosphate hardening solution (H^sub 3^PO^sub ^4) with a pH of 5.6 and a liquid/powder ratio of 0.4 ml/gm. The reason for mixing equimolar TTCP and DCPA was to maintain an overall Ca/P ratio of 1.67 (the theoretical Ca/P of hydroxyapatite). The formation of hydroxyapatite was considered to be the major mechanism for the setting of the CPC.8,9

This cement could be easily shaped in its paste form during surgery and also could be easily injected into a cavity using a syringe without requiring an open passageway through the tissues. The effective working time and setting time of this nd-CPC were found to be 13 and 17 minutes, respectively, such values being considered to be appropriate for most surgical applications.

Mechanical testing of the interfacial healing tissue. The Instron material testing machine model #1322 (Instron, Canton, Massachusetts) featuring a 50 kg load cell was used to detect the maximal tensile strength of the healing tissue. The test samples were dissected to the level of a bone-tendon-bone model, the bones of which were mounted at each end of the tubular mounts with cross pins and resin. The femur was fixed at 45° of flexion in order to align the bone tunnel along the direction of the testing force. Before beginning the stress loading, the suture fixing the grafted tendon to the LCL was removed so that the interface tissue would be the only material to be exposed to the pulling force. The original attachment of the grafted semitendinosus tendon to the tibia was allowed to remain. It was anticipated that the failure point would be situated at the site of the femoral bone tunnel. The tensile force was applied at a rate of displacement of 5.0 mm/s until the point was reached at which the tendon was pulled out of the femoral bone tunnel. The maximal level of tensile strength corresponding to this point was thus determined and the data were analysed statistically by the Wilcoxon signed-rank test.

Histological study of the interfacial healing tissue. Initially, the dissected knees were fixed in a neutralised formalin solution for a period of 72 hours after which the ossified material was decalcified for a period of one week using a mixed solution containing 20% sodium citrate and 50% formic acid. They were then embedded in paraffin and were sectioned perpendicular to the bone tunnel. The tissue was mounted on glass slides, deparaffinised and stained with haematoxylin and eosin for subsequent light microscopy.

Results

All the participating test rabbits tolerated the reconstructive procedure acceptably well. No wound infections were seen and no loss in body-weight was noted.

Histological and gross findings

One-week specimens. Histological examination of the CPC group (Fig. 1) showed that most of the tendon-bone interface was filled with CPC, although thin layers of fibrous tissue were observed between CPC fragments and adjacent to the surface of both the grafted tendon and the bone tunnel. In the non-CPC group, there was a collection of loose tissue in the interface consisting mainly of fibrous tissue.

Three-week specimens. Histological examination showed many growing bone islands within the CPC material some of which had formed directly on the surface of the bone and the tendon. There appeared to be a continuity between the bone and tendon. The healing tissue of the non-CPC group showed an increased production of extracellular collagen fibres which were distributed in a circular orientation around the grafted tendon. Those adjacent to the grafted tendon had an irregular orientation and were interwoven with the fibres of the grafted tendon. Collagen fibres attached to the bone tunnel were observed only occasionally.

Six-week specimens. Histological examination showed that the interface between the bone tunnel and grafted tendon was almost filled by new bone (Fig. 2). The fibres of the grafted tendon appeared to be firmly anchored onto the new growing bone. Some portions of the collagen fibres in the interface had matured to become organised into bundles.

Twelve-week specimens. Histological examination showed that most portions of the bone-tendon interface appeared to have been filled by new bone. Continuity between the collagen fibres of the grafted tendon and surrounding bone was seen. In the non-CPC group no formation of new bone was seen in the interfacial gap and even the native bone trabeculae appeared to have become thinner. An accumulation of adipose cells was found in the interface and in the bone-marrow space. These findings suggested an osteoporotic change.

Twenty-four-week specimens. Histological examination showed that the bone-tendon interface appeared to have healed completely as a result of bone ingrowth and continuity between the collagen fibres of the grafted tendon (Fig. 3). The surrounding bone had been remodelled and resembled Sharpey's fibres.

A layer of newly-formed lamellar bone was observed in the bone tunnel.

Mechanical testing. Six specimens from the CPC group and six from the non-CPC group were tested for the mechanical strength of the bone-tendon union at both one and two weeks. All the specimens failed the test as a result of pullout of the tendon from the femoral bone tunnel. At one week in the CPC group the mean maximal tensile strength of the union was 6.505 ± 1.333 N and in the non-CPC group 2.048 ± 0.950 N (Table I) (Wilcoxon signed-rank test, Z = -2.207, p = 0.027, p

Discussion

The autogenous bone-patellar tendon-bone graft is the most widely used graft for reconstruction of cruciate ligaments. Between 10% and 40%1,6,10,11 of patients report chronic anterior knee pain after such reconstruction. Permanent weakening of the quadriceps by about 10%12,13 is not unusual and kneeling may be uncomfortable. Patellar fractures have been reported in approximately 2% of patients.9,14-16 The quadrupled hamstring tendon graft has attracted wide popularity.17-22 The initial fixation and early healing appear to be inherent weaknesses of the hamstring-graft procedure, and therefore most surgeons, recommend a cautious programme of physiotherapy for periods of up to eight weeks.11,23

A method of augmenting the healing of the tendon to bone may improve the clinical results. The use of a fresh periosteal autograft24 and/or the administration of specific bone morphogenetic proteins25,26 have been reported.

In 1983, Brown and Chow27 reported that the application of a mixture of tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) powders in a diluted phosphate-containing solution led to the formation of hydroxyapatite. Despite its many advantages, there are still some practical problems associated with the use of CPC. These include the prolonged setting time of the material and its potential for dispersion on early contact with blood or aqueous media, substantially limiting its use for bone repair and reconstruction augmentation. Recently, a non-dispersive, faster-setting and high-strength TTCP/ DCPA-based CPC material has been developed in one of the author's (JHCL) laboratories (The Centre for Biological Materials Research at the National Cheng-Kung University). A comparison between this non-dispersive CPC (nd-CPC) and a conventional TTCP/DCPA-based CPC (c-CPC) has been conducted and has shown that during immersion testing, there was a substantial difference in dispersion behaviour between the conventional CPC and the nd-CPC. The latter did not disperse in aqueous solution but even if it did it was in a paste form. Conventional CPC quickly disintegrated in a simulated body fluid even after having set. The nd-CPC material has a higher rate of formation of apatite and also a denser structure than conventional CPC especially in the early stages. Relatively high levels of strength are achieved at an early stage of curing even when it is subjected to immersion in an aqueous medium. The 20-minute compression strength (CS) and diametral tensile strength (DTS) of this new material were relatively high (41.6 and 8.1 Mpa, respectively). The maximum levels (CS = 103 MPa and DTS = 16.3 Mpa) were attained after four to eight days from the time of immersion in a simulated body fluid.8,9

We have shown that the use of CPC augments healing at the tendon-bone interface in this rabbit model as seen histologically by increased bone ingrowth into the tendon and mechanically with increased tensile strength on pull-out tests. The development of new forms of CPC with greater strength may further enhance the process.

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

References

1. Grana WA, EgIe DM, Mahnken R, Goodhart CW. An analysis of autograft fixation after anterior cruciate ligament reconstruction in a rabbit model. Am J Sports Med 1994;3:344-51.

2. Magen H, Howell S, Hull M. Structural properties of six tibial fixation methods for anterior cruciate ligament soft tissue grafts. Am J Sports Med 1999;27:35-43.

3. Kernwein G, Fahey J, Garrison M. The fate of tendon, fascia and elastic connective tissue transplanted into bone. Ann Surg 1938; 108:285-90.

4. Kernwein GA. A study of tendon implantations into bone. Surg Gynec And Obstet 1942:75:794-6.

5. Rodeo SA, Arnoczky SP, Torzilli RA, Hidaka C, Warren RF. Tendon healing in a bone tunnel, a biomechanical study and histological study in the dog. J Bone Joint Surg [Am] 1993;75-A: 1795-803.

6. Arnoczky SP, Torzilli PA, Warren RF, Alien AA. Biologic fixation of ligament protheses and augmentation: an evaluation of bone ingrowth in the dog. Am J Sports Med 1988;16:106-12.

7. Brown WE, Epstein EF. Crystallography of tetracalcium phosphate. J Research Nat Bureau Standards - A. Physics and Chemistry 1965;69-A: 547-51.

8. Chen WC. Ju CP, Chern Lin JH. Effect of heat treatment on compressive strength and setting behavior of TTCP/DCPA-derived calcium phosphate cement. J Mat Sci Lett 2002:21:1583-5.

9. Chen WC, Lin JH, Ju CP. Transmission electron microscopic study on setting mechanism of TTCP/DCPA-derived calcium phosphate cement. J Biomed Mater Res 2003;64-A:664-71.

10. McCarroll JR.Fracture of the patella during a golf swing following reconstruction of the anterior cruciate ligament: a case report. Am JSports Med 1983; 11:26-7.

11. Forward AD, Cowan RJ. Tendon suture to bone: an experimental investigation in rabbits. J Bone Joint Surg [Am] 1963;45-A:807-23.

12. Rosenberg TD, Franklin JL Baldwin GN, Nelson KA. Extensor mechanism function after patellar tendon graft harvest for anterior cruciate ligament reconstruction. Am J Sports Med 1992:20:519-25.

13. Shelbourne KD, Trumper RV. Preventing anterior knee pain after anterior cruciate ligament reconstruction. Am J Sports Med 1997:25:41 -7.

14. Christen B, Jakob RP. Fractures associated with patellar ligament grafts in cruciate ligament surgery. J Bone Joint Surg [Br] 1992;74-B:617-19.

15. Papageorgiou CD, Kostopoulos VK, Moebius EG, et al. Patellar fractures associated with medial-third bone-patellar tendon-bone autograft ACL reconstruction. Knee Surg Sports Traumatol Arthrose 2001;9:151-4.

16. Simonian PT, Mann FA, Mandt PR. Indirect forces and patella fracture after anterior cruciate ligament reconstruction with the patellar ligament: case report. Am J KneeSurg 1995:8:60-5.

17. Brown C, Steiner M, Carson E. The use of hamstring tendons for anterior cruciate ligament reconstruction: technique and results. Clin Sports Med 1993:12:723-56.

18. Cooley VJ, Deffner K, Rosenberg TD. Quadrupled semitendinosus anterior cruciate ligament reconstruction: 5-year results in patients without meniscus loss. Arthroscopy 2001:17:795-800.

19. Corry I, Webb J, Clingeleffer A, Pinczewski LA. Arthroscopic reconstruction of anterior cruciate ligament: a comparison of patellar tendon autograft and four-strand hamstring autograft. Am J Sports Med 1999:27:444-54.

20. Marder RA, Raskind JR, Carroll M. Prospective evaluation of arthroscopically assisted anterior cruciate ligament reconstruction: patellar tendon versus semitendinosus and gracillis tendons. Am J Sports Med 1991:19:478-84.

21. Nebelung W, Becker R, Merke) M, Ropke M. Bone tunnel enlargement after anterior cruciate ligament reconstruction with semitendinosus tendon using Endobutton fixation on the femoral side. Antroscopy1998:14:810-15.

22. Siegel M, Barber-Westin SD. Arthroscopic-assisted outpatient anterior cruciate ligament reconstruction using the semitendinosus and gracillis tendons. Arthroscopy 1998:14:268-77.

23. Clark R, Olsen R, Larson B. Cross-pin femoral fixation: a new technique for hamstring anterior cruciate ligament reconstruction on the knee. Arthroscopy 1998:14:258-67.

24. Ohtera K, Yamada Y, Aoki M, Sasaki T, Yamakoshi K. Effects of periosteal wrapped around tendon in a bone tunnel: a biomechanical and histological study in rabbits. Crit Rev Biomed Eng 2000:28:115-18.

25. Andersen K, Seneviratne AK, Izawa K, et al. Augmentation of tendon healing in an intraarticular bone tunnel with use of a bone growth factor. Am J Sports Med 2001:29:689-98.

26. Rodeo SA, Suzuki K, Deng XH, Wozney J, Warren RF. Use of recombinant human bone morphogenetic protein-2 to enhance tendon healing in a bone tunnel. Am J Sports Med 1999:27:476-88.

27. Brown WE, Chow IC. A new calcium phosphate setting cement (abstract). J Am Dental Research 1983:62:672.

Y.-C. Tien, T.-T. Chih, J.-H. C. Lin, C.-P.Ju, S.-D. Lin

From Kaohsiung Medical University, Kaohsiung, Taiwan

* Y.-C. Tien, MD

* S.-D. Lin, MD

Department of Orthopaedic Surgery, Kaohsiung Medical College, 100 Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan.

* T.-T. Chih, PhD

Fooyin Institute of Technology, 151 Chin-Hsueh Road, Ta-Liao Kaohsiung, Taiwan.

* J.-H.C. Lin, PhD

* C.-P. Ju, PhD

National Cheng-Kung University, 1 Ta-Hsueh Road, Tainan, Taiwan.

Correspondence should be sent to Dr S.-D. Lin.

©2004 British Editorial Society of Bone and Joint Surgery

doi:10.1302/0301-620X.86B7. 14578 $2.00

J Bone Joint Surg [Br] 2004;86-B:1072-6.

Received 30 April 2003; Accepted after revision 8 December 2003

Copyright British Editorial Society of Bone & Joint Surgery Sep 2004
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