The results of the present study show that there is positive chemotactic activity for a progenitor cell population, specifically MRL blastema cells, within the remodeling ECM scaffold material excised from Achilles tendons during the early stages of in vivo degradation and remodeling. The data showed that the degree and temporal pattern of the chemotactic activity is different in MRL/MpJ versus C57BL/6 mice.
The increased migration of progenitor cells toward the remodeling UBM–ECM scaffold in both mouse strains is likely the result of the release of soluble growth factors and matricryptic peptides that form during degradation. ECM scaffolds are known to contain various growth factors that retain their activity even after prolonged shelf life, including TGF-β, b-FGF, and VEGF.39–42
The degradation of ECM scaffolds in vivo
begins immediately after implantation43,44
and is likely associated with the release of both intact proteins and cryptic fragments of parent molecules. It cannot be determined with certainty the extent to which the recruitment of progenitor cells shown in the present was due to soluble growth factors or matricryptic peptides. Recent studies have shown that in vitro
degradation products of UBM–ECM scaffolds are chemotactic for human multilineage progenitor cells and MRL-blastema cells, the cells used in the present study.26
It is unknown whether degradation products formed following the in vivo
degradation process in the ECM-T remodeling tendons are of the same biochemical composition as those formed in vitro
by Li et al
and Reing et al
There is a difference in the chemotactic activity that was measured from the acellular ECM of the scaffold material compared to that measured for the autologous tendon tissue or the normal tendon. One possible explanation for this difference is that the decellularized UBM–ECM scaffold (by definition) lacks any intact cellular component at the time of implantation. In contrast, it is likely that cells in the autologous tendon graft are driven toward apoptosis or necrosis as a result of surgical devascularization and release of proinflammatory mediators. Recent studies that compared the host response to an ECM scaffold and an autologous muscle tissue graft for abdominal wall repair in a rat model showed that the remodeled autologous tissue prompted a proinflammatory macrophage phenotype by 2 weeks after implantation, and the long-term outcome was scar tissue formation.20,45,46
In contrast, the remodeled acellular ECM promoted an anti-inflammatory macrophage phenotype, which resulted in site-specific regeneration of muscle tissue.
A second possible explanation for differences in chemotactic activity between the UBM–ECM and autologous tendon involves the inherent differences between the ECM of tendons and the ECM of urinary bladder. The tendon is a metabolically stable tissue with limited collagen turnover, which allows it to maintain its mechanical strength. Due to the necessity for rapid healing, the ECM of the urinary bladder may have an evolutionary advantage over organs that remodel or heal more slowly. Furthermore, there may be fewer and different growth factors present within tendon ECM. Although several studies have isolated ECM from tendons,47–50
there are no reports of in vivo
remodeling of the harvested tendon ECM.
It is noteworthy that the ECM-T tendons explanted from the MRL mice showed greater chemotactic activity than ECM-T tendons explanted from the C57BL/6 mice at day 3. MRL mice have been described as having rapid healing and enhanced regenerative capability not found in most mammalian species.37,51,52
The regenerative capacity of the MRL mouse was first realized when it was discovered that they spontaneously healed critical-sized ear punches without scar tissue formation,38
although this finding appears to depend on the location and severity of the injury.51,53
When regeneration occurs, the formation of multiple tissue types is associated with a blastema-like structure. The blastema-like structure is the source of the MRL-B cells that were used in the in vitro
assay in the current study originate. Other tissues in the MRL mouse that may have regenerative capability include the heart, brain, and spinal cord.52
The MRL mouse has also been reported to present an abnormal immune system. The relationship of the healing properties and differences in the immune system differences to the finding of our study are unknown and beyond the scope of the manuscript.
It has been shown that MRL mice have increased expression and activity of MMP-2
in the early stages of healing of an ear punch as compared to C57BL/6 mice.54
MRL mice have also been shown to more aggressively degrade basement membrane forms of ECM,54
which is known to be a component of UBM–ECM.36
It is possible that increased expression and activity of MMPs in the MRL/MpJ strain may lead to more rapid degradation than occurs in the C57BL/6 mouse, and thus an earlier release of chemotactic degradation products. Therefore, if indeed the release of matricryptic peptides were the main factor responsible for the recruitment of progenitor cells, it would be expected that the C57BL/6 would display a delayed response. The degradation of the ECM scaffolds was not quantitatively measured in the current study, so additional work will be required to determine whether the degradation profiles of the ECM scaffolds differ for the two strains.
The ECM did not appear to provide positive recruitment of progenitor cells at 14 days after implantation, regardless of the strain. Certainly, large portions of the scaffold are still present beyond 7 days, and this scaffold material continues to remodel. However, there is also an active host cellular response that is characterized by a phenotypically different population of cells, which may in turn affect the recruitment of progenitor cells. Therefore, the tissue tested from the 14-day explants represented a mixture of xenogeneic scaffold, newly deposited host tissue, and an abundance of host cells. Day 14 is the point at which the level of cellularity typically peaks during the remodeling of an ECM scaffold, after which point the degree of cellularity gradually decreases. The same mechanisms that explain this decrease in cellularity may also be responsible for the decreased recruitment of progenitor cells.
There are several limitations of the current study. First, the source of progenitor cells (MRL-blastema) chosen for the current study is native to one of the strains used for the in vivo
implantation, specifically the MRL/MpJ.37,38,55
However, the remodeled UBM–ECM harvested from the surgical site in both mouse strains showed more chemotactic potential than the remodeled autologous tendon. Bone marrow–derived mononuclear cells would have been an interesting population to examine since it is known that bone marrow–derived cells are recruited to the site of ECM remodeling and become part of the tissue, but the specific population of marrow-derived cells to evaluate would have been an arbitrary choice. The specific populations of cells recruited to the site of ECM remodeling are an area of current interest. Finally, this study does not specifically identify the cell-specific enzymes that contribute to scaffold degradation in the Achilles tendon model.