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Application of layered poly (L-lactic acid) cell free scaffold in a rabbit rotator cuff defect model
© Inui et al; licensee BioMed Central Ltd. 2011
Received: 13 April 2011
Accepted: 2 December 2011
Published: 2 December 2011
This study evaluated the application of a layered cell free poly (L-lactic acid) (PLLA) scaffold to regenerate an infraspinatus tendon defect in a rabbit model. We hypothesized that PLLA scaffold without cultivated cells would lead to regeneration of tissue with mechanical properties similar to reattached infraspinatus without tendon defects.
Layered PLLA fabric with a smooth surface on one side and a pile-finished surface on the other side was used. Novel form of layered PLLA scaffold was created by superimposing 2 PLLA fabrics. Defects of the infraspinatus tendon were created in 32 rabbits and the PLLA scaffolds were transplanted, four rabbits were used as normal control. Contralateral infraspinatus tendons were reattached to humeral head without scaffold implantation. Histological and mechanical evaluations were performed at 4, 8, and 16 weeks after operation.
At 4 weeks postoperatively, cell migration was observed in the interstice of the PLLA fibers. Regenerated tissue was directly connected to the bone composed mainly of type III collagen, at 16 weeks postoperatively. The ultimate failure load increased in a time-dependent manner and no statistical difference was seen between normal infraspinatus tendon and scaffold group at 8 and 16 weeks postoperatively. There were no differences between scaffold group and reattach group at each time of point. The stiffness did not improve significantly in both groups.
A novel form of layered PLLA scaffold has the potential to induce cell migration into the scaffold and to bridge the tendon defect with mechanical properties similar to reattached infraspinatus tendon model.
Rotator cuff tear is a common problem that may cause chronic pain with functional disability. If intolerable pain or functional disability persists, surgical treatment can be considered. Excellent outcomes have been reported with arthroscopic rotator cuff repair in small or medium sized tears , but large or massive rotator cuff tears are challenging for surgeons . Surgical procedures are available for repair of massive rotator cuff tears including musculotendinous transfer  and patch grafting using fascial tissue  or synthetic materials [5–8], but these techniques have disadvantages. Tendon transfer is not an anatomical reconstruction and patch grafting using fascial tissue requires sacrifice of normal tissue. Using extracellular matrix derived from animals such as porcine small intestinal submucosa can cause an immune reaction and carry the risk of zoonoses resulting from xenotransplantation . Nonabsorbable synthetic materials have been used , but foreign body reaction and bone erosion has been reported several years after surgery .
In tendon regeneration filed, tissue engineering using scaffolds, cultured cells and growth factors has been showing great advantage . In a controlled laboratory study on rabbit model, fibroblast-seeded scaffolds showed better results compared to the scaffold alone in rotator cuff regeneration . However, these methods require 2-stage surgeries; a first surgery to harvest cells and a second to transplant them. Since 2-step surgeries are considered invasive, less invasive methods need to be developed. If the scaffold could induce cells from the surrounding tissues, pre-expansion of the cells for the transplantation would not be required. The authors assessed a double layered form of bioabsorbable poly (L-lactic acid) (PLLA) fabric which have smooth surface on one side and pile-finished surface on the other side, and reported its potency for regenerations of tendons and ligaments in a rabbit model . The author hypothesized that PLLA scaffold without cultivated cells would lead to regeneration of tissue with mechanical properties similar to reattached infraspinatus without tendon defects. In the current study, application of the layered PLLA scaffold was evaluated for the regeneration of rotator cuff tears in a rabbit model.
All the animal experiments were approved by the Animal Research Committee.
Preparation of Layered PLLA Scaffold
After the macroscopic examination, the infraspinatus tendon humeral head complex was fixed in 4% paraformaldehyde, decalcified with 0.25 mol/l ethylenediaminetetraacetic acid in a phosphate buffered saline (pH 7.5), and embedded in paraffin. Sagittal sections (7 μm) were cut through the entire thickness of the defect, and stained with hematoxylin eosin (HE) then examined by light microscopy. For immunohistochemical analysis of type I and III collagens, deparaffinized sections were digested with proteinase (Dako Cytomation, Inc., CA, USA) for 10 minutes and treated with 3% hydrogen peroxide (WAKO Pure Chemical Industries, Ltd., Osaka, Japan) to block endogenous peroxidase activity. The sections were incubated with a 1:50 dilution of goat anti-type I or III collagen antibodies (Southern Biotechnology Associates Inc., AL, USA) at 4°C overnight and subsequently treated with peroxidase labeled anti-goat immunoglobulin (Histofine Simplestain max PO(G), Nichirei Bioscience, Tokyo, Japan) at room temperature for 30 minutes. The signal was developed using peroxidase substrate 3, 3-diaminobenzide (Histofine Simplestain DAB Solution, Nichirei Bioscience, Tokyo, Japan) and the sections were examined microscopically.
The infraspinatus tendon humeral head complex was harvested from each shoulder and all soft tissue except for the infraspinatus tendon was resected by sharp dissection. The humerus was potted in specially designed devices using polymethylmethacrylate (PMMA) resin and a specially designed device clamped the end of each tendon. The complex was placed vertically to a tensile sensor (AG-I SHIMAZU Co, Kyoto, Japan). Before the tensile test was conducted, the infraspinatus tendon humeral head complex was preconditioned with a static preload of 0.5 N for 5 minutes, followed by 10 cycles of loading and unloading at strain amplitude of approximately 0.5% at a rate of 20 mm per minute. Immediately after preconditioning, the ultimate load to failure was recorded in uniaxial tension at 20 mm per minute . The ultimate load to failure and the stiffness were measured from load-deformation curve. Mechanical properties of normal infraspinatus tendon and scaffold itself were evaluated in the same manner.
The paired t test was used to compare the mechanical properties of PLLA scaffold repair with the reattached tendon. Dunnett's test was used to compare the mechanical properties at each time point. A significance level of p < .05 was used. All data are presented as mean (SD).
Mechanical Properties of Regenerated Tissue
Several synthetic materials have been previously used in rotator cuff regeneration. Implantation of polytetrafuluoroethylene (PTFE) patch grafts showed satisfactory functional results in 23 out of 25 patients with massive rotator cuff tears . However, reconstruction of rotator cuff defects with PTFE in a canine model showed foreign body reaction at 12-24 weeks after surgery and bone resorption at the insertion of the PTFE into the bone . Chitin fabric used as a scaffold in rabbit rotator cuff tears resulted in regenerated tissue, mainly composed by type III collagen . Finally, poly glycolic acid (PGA) sheets used as a scaffold showed a well-arranged fibrocartilage layer in regenerated tendon insertions .
All the scaffolds previously used for rotator cuff tears had a uniform structure [5–8, 10, 11]. The authors previously reported the potency of double layered PLLA fabric which has smooth surface on one side and pile-finished surface on the other side for tendon tissue engineering. In its pile-finished side, cell migration was observed. On the other hand, smooth side showed less adhesion. The double layered fabric maintained its mechanical property compared with plain-woven fabric . In present study, the authors chose this PLLA fabric as the scaffold material for rotator cuff repair study. A substantial amount of spindle shaped cells migrated inside the layered scaffold. The authors also noted several giant polynuclear cells indicative of an inflammatory reaction.
PLLA fiber is an organic polymer from L-lactate that dissolves in H2O and CO2. PLLA was used as a material for human orthopedic implants  and as synthetic material in ligament or tendon tissue engineering research . An in vitro study that compared PGA, poly-L-lactate-epsilon-caprolactone (PLC), and PLLA scaffolds concluded that PLLA and PLC scaffolds provided the best material for tendon regeneration because of slow absorption rates and better mechanical properties . Augmentation of rotator cuff repair with a PLLA scaffold provided sufficient initial strength in a sheep model , but the byproducts of PLLA degradation might have inhibited the tendon from healing to bone . In present study, the ultimate failure load of the scaffold group increased over time and was not different from normal infraspinatus tendon 8 weeks after surgery. No difference was observed between the scaffold group and reattachment control group, indicating that the implantation of the layered PLLA scaffold in rotator cuff defect had same strength as reattachment. Thus the authors could confirm our hypothesis that PLLA scaffold without cultivated cells would lead to regeneration of tissue with mechanical properties similar to reattached infraspinatus without tendon defects. However, the stiffness was weaker in the scaffold group than normal infraspinatus tendon, indicating that the regenerated tissue had not matured as a normal tendon. While type I collagen is prominent in the normal rotator cuff tendon, type III collagen is expressed during the tendon healing process and its levels decrease as the tendon matures [19, 20]. Therefore, if the regenerated tissues mainly consist of type III collagen, the mechanical property of these tissues is presumably inferior to the native tissues [21–23]. Dominant expression of type III collagen was observed, which may have been caused by the low absorption rate of the PLLA fiber that can lead to inferior stiffness properties compared with normal infraspinatus tendon.
There were some limitations in this study. Firstly, the anatomy of a rabbit's shoulder differs from the human rotator cuff because the short rotator muscles of rabbits do not form a rotator cuff as in humans. Secondly, the rabbit model in present study differs from chronic rotator cuff injury model. Thirdly, the PLLA scaffold can induce an inflammatory reaction. Clinical studies showed that a systemic or local reaction might arise. Although the degradation products of PLLA are natural metabolites, they are acidic [14, 24]. The absorption rate of the scaffold must be taken into consideration and long-term follow up is required to confirm results.
A novel form of layered PLLA scaffold was used to repair rotator cuff defect in a rabbit model. This layered form of the PLLA scaffold has the potential to induce cell migration and to fill the gap between the bone and the tendon stump, with mechanical properties similar to reattached infraspinatus.
This study was supported in part by the ministry of Education, Culture, Sports, Science and Technology-Japan grants No.197912030 for aiding scientific researchers.
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