Role of xenogenous bovine platelet gel embedded within collagen implant on tendon healing: an in vitro and in vivo study

Abstract

Surgical reconstruction of large Achilles tendon defects is demanding. Platelet concentrates may be useful to favor healing in such conditions. The characteristics of bovine platelet-gel embedded within a collagen-implant were determined in vitro, and its healing efficacy was examined in a large Achilles tendon defect in rabbits. Two cm of the left Achilles tendon of 60 rabbits were excised, and the animals were randomly assigned to control (no implant), collagen-implant, or bovine-platelet-gel-collagen-implant groups. The tendon edges were maintained aligned using a Kessler suture. No implant was inserted in the control group. In the two other groups, a collagen-implant or bovine-platelet-gel-collagen-implant was inserted in the defect. The bioelectricity and serum platelet-derived growth factor levels were measured weekly and at 60 days post injury, respectively. After euthanasia at 60 days post injury, the tendons were tested at macroscopic, microscopic, and ultrastructural levels, and their dry matter and biomechanical performances were also assessed. Another 60 rabbits were assigned to receive no implant, a collagen-implant, or a bovine-platelet-gel-collagen-implant, euthanized at 10, 20, 30, and 40 days post injury, and their tendons were evaluated grossly and histologically to determine host-graft interactions. Compared to the control and collagen-implant, treatment with bovine-platelet-gel-collagen-implant improved tissue bioelectricity and serum platelet-derived growth factor levels, and increased cell proliferation, differentiation, and maturation. It also increased number, diameter, and density of the collagen fibrils, alignment and maturation of the collagen fibrils and fibers, biomechanical properties and dry matter content of the injured tendons at 60 days post injury. The bovine-platelet-gel-collagen-implant also increased biodegradability, biocompatibility, and tissue incorporation behavior of the implant compared to the collagen-implant alone. This treatment also decreased tendon adhesion, muscle fibrosis, and atrophy, and improved the physical activity of the animals. The bovine-platelet-gel-collagen-implant was effective in neotenon formation in vivo, which may be valuable in the clinical setting.

Keywords: Tendon; biomechanics; collagen; host graft interaction; platelet gel; tissue engineering; ultrastructure.

Figure 1

(a) Preparation of the BPG embedded within CI. Each arrow shows a subsequent step. (b) An SEM of the BPG-CI. The platelets (arrows) well attached to the collagen fibers (arrow head). (c) The activated platelets (thick arrow) attached to the fibrin matrix (arrow head) and the collagen fibers (thin arrow). (d) An SEM of the activated and accumulated platelets. (e) Cytologic appearance of the platelets after the second centrifugation. (f) The cytologic section of the bovine PRP by polarized microscopy. (g) The TEM of the bovine platelets after the second centrifugation. Scale bar: (b) 1 µm; (c) 750 nm; (d) 500 nm; (e) 3 µm; (f) 12 µm; (g) 630 nm. (A color version of this figure is available in the online journal.)

Figure 2

Surgical operation. (a) Surgical site and preparation method. (b) Skin incision and exposure of the Achilles tendon apparatus (ATA). (c) The black arrows point to the gastroc. soleus muscle proximally and calcaneal tuberosity distally. The red arrow points to the ATA. The blue arrows point to the segment to be removed, and the yellow arrow points to the tibialis posterior tendon. (d and e) Two cm of the ATA was measured and removed by sharp dissection. (f) A modified Kessler suture was anchored at the edges of the remaining tendon. (g and h) The CI and BPG-CI were inserted. (A color version of this figure is available in the online journal.)

Figure 3

In vitro characteristics of the bovine platelets. (A color version of this figure is available in the online journal.)

Figure 4

Live/dead cell assay, immunoflorescent microscopy, and SEM of the cultured fibroblasts on CI, BPG-CI, and BPG. The BPG increased cellular migration, proliferation, and matrix production in vitro. (A color version of this figure is available in the online journal.)

Figure 5

The BPG significantly reduced DTEC and increased TRDTEC of the ITTC-PGs and approximated them to the normal values in comparison to the controls. At 60 DPI, those animals treated with BPG-CI showed significantly higher serum PDGF level than controls. (A color version of this figure is available in the online journal.)

Figure 6

(a) ICTs (no implant). (b) ITTCs. (c) ITTC-PGs. (d) NCTs. At 60 DPI, the PG considerably decreased tendon hyperemia, peritendinous adhesions, muscle fibrosis, and atrophy and it increased the homogeneity and density of the newly formed fibrous connective tissue in the defect area when compared to the controls. (A color version of this figure is available in the online journal.)

Figure 7

Histopathological evaluation of host-graft interaction at various stages of tendon healing. (a to d) ITTCs at 10, 20, 30, and 40 DPI. (e to h) ITTC-PGs at 10, 20, 30, and 40 DPI, respectively. At 10 DPI, in the ITTCs (a), the inflammatory cells (I) were accumulated in the peripheral parts of the collagen implant (CI) and were degrading it. The peripheral new granulation tissue (PNGT) has covered the CI at this stage. In contrast (a), in the ITTC-PGs (e), the inflammatory cells were evenly distributed throughout the implant (I + CI) at 10 DPI. At 20 DPI, some parts of the CI were invaded by high accumulation of inflammatory cells (b) but some collagen remnants (CR) remained with no inflammatory reaction. In contrast to the ITTCs, in the ITTC-PGs, the CR were free of inflammatory cells accumulation (f). The newly developed connective tissue filled the free spaces between the CR. At 30 DPI, in the ITTCs, some parts of the CR were infiltrated by the tenoblasts and were accepted as parts of the neotenon, but the newly regenerated tissue which surrounded the CR were immature in nature, consisting of immature vessels and low collagen density (C). In contrast to the ITTCs, in the ITTC-PGs, the CR were well infiltrated by the host tenoblasts and the newly regenerated tissue which covered the CR was a mature connective tissue (MCT) consisting of aligned collagen fibers and a combination of tenoblast and tenocytes (g). At 40 DPI, in the ITTCs, some other CR were also degraded and replaced by the new connective tissue so that there were two distinct areas of the mature (MCF) and immature (ICF) collagen fibers (d). In contrast to the ITTCs, most of the new tendon, in the ITTC-PGs, was filled with the mature collagen fibers (MCF) at this stage (h). (A color version of this figure is available in the online journal.)

Figure 8

Differential cell counts in the histology sections of the injured healing tendons at 60 DPI. In each group: n tendon samples = 10; n histologic sections = 5; n histologic field = 5; totally 250 field. The results were expressed as mean ± standard deviation. (A color version of this figure is available in the online journal.)

Figure 9

Histopathologic characteristics of the injured and normal tendons at 60 DPI. (a), (e), (i): ICTs; (b), (f), (j) ITTCs; (c), (g), (k): ITTC-PGs; (d), (h), (l): NCTs. (a to d) Longitudinal sections of the tendons, low magnification view (Scale bar = 50 µm); (e to h) longitudinal sections of the tendons, high magnification view (Scale bar = 12.5 µm); (i to l) the histologic sections of the gastroc. soleus muscle (Scale bar = 50 µm). (a to d) The long thin arrows show the orientation of the collagen fibers. At 60 DPI, only a loose areolar connective tissue, consisting of immature tenoblasts and low-density collagen material, had formed in the ICTs (a and e). At this stage, a tendon-like tissue was formed in the ITTCs and the collagen fibers were well developed and the cells were a combination of immature (arrows head) and mature tenoblasts. However, the collagen fibers were not completely aligned because they laid at various directions (b and f). In contrast to the ITTCs, the newly developed tendon-like tissue in the ITTC-PGs was more mature, consisting of highly aligned collagen fibers and a combination of mature tenoblasts (thick arrows) and tenocytes (thin arrows) laid along the direction of the collagen fibers (c and g). In NCTs, large collagen fibers were oriented unidirectionally (arrows) and few tenocytes organized along these aligned collagen fibers (d and h). The ICTs showed severe muscle atrophy and fibrosis so that the muscle fibers (arrow) were small and were severely infiltrated by fibrous connective tissue (i). The CI reduced muscle fibrosis and atrophy (j). The BPG-CI considerably increased the muscle fiber size (k) when compared to the NCTs (l). Color staining: H&E. (A color version of this figure is available in the online journal.)

Figure 10

TEM of the injured and normal tendons at 60 DPI. (a to d) Transverse sections of the injured and normal tendons. (e to h) Longitudinal sections of the injured and normal tendons. (a and e) ICTs. (b and f) ITTCs. (c and g) ITTC-PGs. (d and h) NCTs. Treatment with CI significantly increased the number, diameter, and density of the collagen fibrils in the ITTCs when compared to the ICTs. However, the collagen fibrils were still unimodal and of small-sized diameter. In contrast to the ITTCs, the ITTC-PGs showed significantly larger diameter of the collagen fibrils so that the large collagen fibrils comparable to the normal tendon (arrows) were formed in these tendons (c). The treated tendons also had aligned collagen fibrils, so that most of the collagen fibrils (arrows) were aligned unidirectionally along the longitudinal axis of tendon. Compared to the ICTs (e), the treated tendons showed continuous collagen fibrils (f and g) while in the ICTs, the collagen fibrils were small-sized and some of them were not continuous. Scale bar: (a): 500 nm, (b) 870 nm, (c) 800 nm, (d) 600 nm, (e) 550 nm, (f) 670 nm, (g) 700 nm, (h) 650 nm. Staining: Lead citrate and uranyl acetate. (A color version of this figure is available in the online journal.)

Figure 11

Biomechanical properties and dry matter content of the injured healing tendons at 60 DPI. Number of tendons in each group = 10. The results were expressed as mean ± standard deviation. (A color version of this figure is available in the online journal.)