Figure 1.
A: Non-aligned electrospun collagen fibers constructed by electrospinning without dual plate device. B: Aligned electrospun collagen fibers were produced, using gap collector. C: Aligned internal architecture of the polymerized collagen fibers in the gel. D: The composition of the collagen implant at a larger magnification. E and F: Transverse section of the collagen implant after crosslinking. Most of the implant is filled with the polymerized collagen fibers while little porosity exists between these fibers. G: Surface of the collagen implant after crosslinking. H–J: Surface of the polydioxanone sheath. Note that there is no porosity in the external and internal architecture of the polydioxanone sheath. K: Circular polydioxanone sheath. L: Polydioxanone plate was melted and wrapped around the collagen implant to produce a collagen-PDS scaffold. PDS sheath could be seen at periphery while the collagen implant is seen in the center.
Figure 2.
Surgical intervention and implantation of the bioimplant.
A: The surgical site and preparation method. B: Skin incision and exposure of the Achilles apparatus. C: Black arrows show the gastro soleus muscle proximally and calcaneal tuberosity distally. The red arrow shows the Achilles apparatus, blue arrows the segment to be removed and the yellow arrow the tibialis posterior tendon. D and E: 2 cm of the Achilles tendon was measured with a digital caliper and removed. F: A modified Kessler Core pattern suture was anchored in the ends of the remaining tendon. G and H: The collagen and collagen PDS implants were inserted. I: The skin over the lesion was closed.
Figure 3.
A: transverse diameter of the lesion (skin, fascia and Achilles apparatus): The diameter of the ICTs significantly increased (P<0.05) at 7 DPI and was elevated at 14 DPI, compared to the NCTs. At 14 to 30 DPI, the diameter of the injured area decreased continually so that it reached its normal value at 30 DPI. From day 30 to 60 DPI, the diameter of the ICTs significantly decreased and was lower than normal value. The diameter of the ITTCs and ITTC-PDSs significantly increased during the first 14 DPI with the peak level at 14 DPI. The measured value of these tendons was significantly higher than the ICTs at 14 DPI. Also, the measured value of the ITTC-PDSs was significantly higher than those of ITTCs at the same stage. From 14 to 60 DPI, the diameter of the injured area of the ITTCs and ITTC-PDSs continually decreased and reached their normal level at 60 DPI. B: Surface temperature of the injured area from the skin over the lesion. The temperature of the ICTs significantly increased during the first 14 days after injury compared to intact tendons. Then after, it decreased to the normal level that was observed in intact tendons, at 20 DPI. The temperature of the ITTCs and ITTC-PDSs significantly increased during the first 14 DPI but the measured value for ITTC-PDSs was significantly higher than those of ITTCs at this stage. The temperature of the ITTCs and ITTC-PDSs, although significantly higher than ICTs at 20 DPI, showed no significant differences between ITTCs and ITTC-PDSs at 20 DPI. At 30 DPI, the measured value for these two last tendons was not statistically different compared to the intact tendons.
Figure 4.
Ultrasonographical characteristics of the tendons.
The ICT shows hypoechogenicity with high amount of peritendinous adhesion that is observed as a hypoechoic pattern in the peritendinous area (A). This tendon also has amputated view under inverted ultrasonography (E) that is characterized with irregular hyper echogenicity together with the anechoic pattern (E). The diameter of the regenerated tissue (Arrows) is low (A) and severe development of peritendinous adhesion is evident (E, arrows head). The ITTC, shows hyperechogenicity (B) together with homogeneity (F) of the echogenic area of the tendon (Arrows). The diameter of the new tendon is characteristically higher than ICT (F vs. E). ITTC-PDS (Arrows) has higher transverse diameter compared to the ITTC (Arrows) and ICT (Arrows). It is also more echogenic and no amputated pattern is observable in this tendon (C). These tendons (B and C) are more uniform than ICTs. The amount of peritendinous adhesion is also decreased in this tendon (C). Intact tendons have a regular pattern with the hyper echogenic texture (D,H). The paratenon can be seen around the Achilles apparatus as two tiny echogenic lines (D,H Arrows head). In an inverted view better judgment can be made on the differences of the tendons.
Table 1.
Peripheral blood profile of the treated and control animals at 10 and 60 days after the injury.
Table 2.
Biochemical findings of the injured tendons at 60 days after the injury.
Figure 5.
Gross pathological changes in the injured tendons.
A–C: ITTC. A: 10, B: 30, C: 60 DPI. D–F: ITTC-PDS. D: 10, E: 30, F: 60 DPI. G–I: ICT. G: 10, H: 30, I: 60 DPI. J: NCT. At 10 DPI, the transverse diameter of the treated lesions is significantly increased compared to the control tendons (A and D vs. G). The implants are surrounded by the newly regenerated fibrous connective tissue at this stage. At 30 DPI, the collagen implant is completely degraded and a new tendon formed (B). The tendon is transparent at this stage (B). Compared to the ITTC, the remnants of the PDS sheath is seen in the injured area of the ITTC-PDSs at 30 DPI (E). Compared to the treated lesions, the control tendon and its gastro soleus muscle are atrophied at this stage and sever hyperemia is seen all over the tendon (B and E vs. H). At 60 DPI, the newly regenerated tendon is matured and is dense. The muscle is less atrophied and less fibrosis is evident in the treated lesions compared to control (C and F vs. I). At 60 DPI, the severity of hyperemia is decreased in the ICTs (I) but it is completely atrophied and its diameter is small. Unlike the ICTs, the transverse diameter of the treated lesions (C and F) is comparable to normal intact tendon (J). Compared to the ITTC, the ITTC-PDS shows less muscle atrophy and no remnants of the PDS sheath is visually evident at gross level (E).
Figure 6.
Host implant interaction (Mechanism of collagen implant absorption and its role on tendon healing).
At 10 DPI the collagen implant absorbed the inflammatory and mesenchymal cells so that three areas can be observed (A). In the first area, collagen implant (C.I.) is unaffected by the inflammatory cells. The second area is characterized by infiltration of the inflammatory cells (I.) and the third area is a newly developed granulation tissue (G.T.). In the second area, the inflammatory cells degrade the collagen implant and the remnants of the collagen implant are seen (B). At 15 DPI, some parts of the collagen implant are degraded by the inflammatory cells but some parts of the collagen implant are unaffected and are called collagen remnant (C.R.). The G.T. is developed between the degraded parts of the collagen implant. From 20 to 60 DPI, the preserved parts of the collagen implant are incorporated with newly developed healing tissue by three mechanisms. Mechanism 1 (D to I) is a progressive degradation in which the unaffected parts of the collagen implant act as a micro scaffold for the newly regenerated connective tissue and align the new tissue along their orientation. They are gradually degraded but not as fast as those parts degraded at inflammatory phase (B). in this mechanism the collagen implant is almost degraded and replaced by the new tendon so that at 60 DPI (I) the new tendon can be seen. Mechanism 2 (J and K) is the rapid absorption of some parts of the collagen implant. In such mechanism some parts of the collagen implant are absorbed at earlier stages (e.g. 10 DPI) and replaced by the new connective tissue which is matured over time. At later stages (e.g. 30 DPI) some other parts of the collagen implant are absorbed and replaced by the newly developed connective tissue so that at 30 (J) to 40 (K) DPI, two types of newly developed mature and immature collagen fibers (M.C.F. and I.C.F.) can be seen. The M.C.F. act as a micro scaffold for the I.C.F. and align them along their longitudinal direction. These immature collagen fibers are more mature in later stages of tendon healing (J vs. K). Mechanism 3 (L and M) is the graft acceptance in which some preserved parts of the collagen implant are infiltrated by the healing fibroblasts and act as a micro scaffold for the newly developed connective tissue. N: Transverse section of the healing tendons. At 60 DPI, most parts of the collagen implant are degraded and replaced by the new tendon. At longitudinal section (O) the new tendon (N.T) is seen and no remnant of the collagen implant is seen, which means the implant is almost degraded and replaced by the newly aligned tendinous tissue. Color staining: H&E.
Figure 7.
A: The remnants of the polydioxanone (PDS) suture knots are shown by arrow. B: The arrow shows a remnant of the PDS suture in the healing tissue. C: Injured control tendon (ICT), 60 DPI: fatty tissue has infiltrated in the defect area so that no tendinous tissue is seen. D: An ICT, 60 DPI: the newly developed collagen fibers are immature, and the tissue is amorphous. E: An injured treated tendon with collagen implant (ITTC), 60 DPI: the collagen fibers are mature and highly aligned. F to H: An injured treated tendon with collagen-PDS implant (ITTC-PDSs), 60 DPI: the collagen fibers are aligned and are highly mature. In the transverse (G) and longitudinal (H) sections, the remnants of the PDS scaffold are seen which cover the new tendon (N.T.). No remnant of the collagen implant is seen and all of the tendinous tissue is newly developed. I: A normal intact tendon. The collagen fibers are highly dense and are aligned. In figures D to F and I the double arrows show the direction of the collagen fibers. J: An ICT, 60 DPI: the cells are immature and randomly distributed in the new tissue. The collagen fibers are highly immature and no characteristic alignment can be seen. K: An ITTC, 60 DPI: most of the cells are mature fibroblast and fibrocytes which are laid along the direction of the newly developed highly aligned collagen fibers. L: An ITTC-PDSs, 60 DPI: the majority of the cells are fibrocyte with few number of mature fibroblast. The cells are highly aligned and laid along the direction of the collagen fibers. M: a gastro soleus muscle of the ICT, 60 DPI. Look, the connective tissue is developed between the atrophic muscle fibers which indicates both fibrosis and atrophy of this muscle. N: a gastro soleus muscle of the ITTC, 60 DPI. A lesser amount of connective tissue is developed between the muscle fibers (arrow) and size of the muscle fibers is larger than M which indicates lower degree of muscle fibrosis and atrophy. O: is a normal (intact) gastro soleus muscle. In a normal muscle, the fibers are larger than M and N and less connective tissue is evident between the fibers.
Figure 8.
Intrinsic and extrinsic mechanisms of tendon healing.
A–C: Transverse section of the skin, fascia and defect area after 60 days post injury (DPI). D–F: Longitudinal section of the skin, fascia and defect area after 60 DPI. A and D: control (defect) tendons. B and E: Injured treated tendons with collagen implant. C and F: Injured treated tendons with collagen-PDS implant. In the control lesions no tendinous structure is formed in the defect area at 60 DPI and the defect is filled with a loose areolar connective tissue. This suggests that the intrinsic mechanism of tendon healing was not effective in producing new tendon in the control group. In the control group a fascia was formed between the skin and defect area and therefore the peritendinous adhesion was well developed. In these tendons, the layers could not be well distinguished from each other and the skin is tightly adhered to the newly regenerated tissue and therefore, these tendons had no function because they cannot move in their subcutaneous space. Implantation of the collagen implant was able to produce a new dense tendinous structure in the defect area and also reduced peritendinous adhesion. These findings suggest that the collagen implant resulted in improving the intrinsic mechanism of tendon healing and was effective in reducing extrinsic mechanism which is responsible for the development of peritendinous adhesions. Therefore, a fascia could be seen between the skin and new tendon but the density and amount of this fascia is lower than those seen in the control lesions at the same stage. Implantation of the collagen-PDS implant significantly reduced peritendinous adhesions as compared to controls and also a new tendon is formed inside the PDS sheath while the collagen implant is replaced by the new tendon. In these tendons no marked peritendinous adhesion is formed around the new tendon but a loose fascia is present just under the skin which is normal. The remnants of the PDS scaffold are present at the periphery of the newly regenerated tendon. These findings suggest that the PDS sheath completely inhibited the extrinsic mechanism of tendon healing and tendon adhesions but had no deleterious effect on the intrinsic mechanism of tendon healing. The right one/third of the figures are inverted to differentiate the structures more comprehensively.
Table 3.
Histopathological findings (number and diameter of different cellular and vascular structures in the histopathologic field).
Figure 9.
Ultrastructural characteristics of the tendons (powered by colored scanning electron microscopy).
ICTs had a lower density of the collagen fibrils (A) and aggregation of this haphazardly oriented collagen fibrils failed to produce the collagen fibers as it was seen in the ITTCs (B), ITTC-PDSs (C) and Intact tendons (D). Compared to the ITTCs (B) the fibers were more differentiated and developed in the ITTC-PDSs (C). At a larger magnification (E–H), the collagen fibrils were randomly distributed in the ICTs and not aggregated as fibers. The diameter and density of the collagen fibrils was low. Unlike ICTs, the fibers were more developed in the treated lesions (F, G) but these fibers were not highly aligned as it can be seen in intact tendons (H). I–L: are the transverse sections of the tendons. In the ICTs the cellularity was high and no diagnostic fibers and fiber bundles formed. The section was only filled with the unorganized collagen fibrils that produce an amorphous structure at lower magnification (I). Fibers and Fiber bundles were differentiated in the ITTCs (J) and ITTC-PDSs (K) and could be distinguished from each other. These fibers compactly aggregated to produce the bundles of fibers. Although these fibers were formed they were not as dense as those we have seen in intact tendons (L). This is because the density and compactness of the collagen fibrils of the ITTCs and ITTC-PDSs were lower than intact tendons. Scale bar: A = 5 µm, B–D = 40 µm, E–G = 15 µm, H = 80 µm, I–L = 25 µm.
Table 4.
Morphological characteristics of the injured tendons at 60 days after injury (Scanning electron microscopy).
Table 5.
Biomechanical properties of the injured and normal contralateral tendons after 60 days of tendon injury.