Unlike salamanders, there is currently no regenerative therapy for limb and digit amputation in humans. While prosthetic technologies have dramatically improved, prosthetics fail to restore quality of life to the pre-injury state. The Badylak laboratory has been pursuing regenerative medicine solutions for functional limb and digit regeneration.
Complex tissue regeneration
The Badylak laboratory has established a non-regenerating mouse model of digit amputation, which is used to study innovative approaches for functional tissue reconstruction, most of which are based upon the inductive effects of extracellular matrix (ECM) (Figure 1) [1,2]. Using this model, we have shown that ECM can positively affect both the recruitment of endogenous stem cells and modulation of the innate immune response.
Figure 1: H&E image of mouse digit, with amputation site indicated in black.
Using ECM derived from a porcine urinary bladder matrix (UBM) that was solubilized by pepsin digestion, amputated mouse digits were treated daily for four days and observed the rapid accumulation of a cluster of primitive cells at the site of injury, including multipotential cells with Sox2 and Sca1 markers (Figure 2A). When isolated, these cells were capable of differentiation along osteogenic, adipogenic, and neuronal pathways in vitro (Figure 2B).
Figure 2: A - In the injured digit, more Sox2+ and Sca1+ cells and more proliferating cells (Ki67+) are present when treated with peptide derived from ECM, as opposed to PBS. B - Isolated cells from the digit tip were shown to be multipotential, differentiating into different cell types in vitro.
The UBM degradation products and solubilized signaling molecules modify the phenotype of macrophages that respond to the digit injury. Using immunofluorescence labeling, a greater ratio of M2, pro-remodeling macrophages to M1, pro-inflammatory macrophages was present at 7 and 14 days after injury. The ability to modulate macrophage phenotype holds significant promise for future tissue remodeling applications. The Badylak laboratory has identified macrophage polarization as a predictor of tissue remodeling outcomes in several clinical applications [3,4].
Figure 3: Quantification of immunofluorescence staining for markers of M1 and M2 macrophages was used to characterize the immune response, which has a predominantly more M2:M1 phenotype than the non-treatment control at 7 and 14 days.
While work in the mouse digit model has been promising, questions remain regarding the mechanism by which UBM recruits these progenitor cells to the site of injury. Additionally, methods to promote differentiation of the recruited multipotential cells are being explored.
Presently, a study is in progress to examine the effect of bioactive ECM molecules as well as factors such as pH, O2 concentration, and electrical potential on the functional remodeling of injured soft tissue in a large animal model. The goal of the study is to identify the optimal microenvironment for tissue remodeling and to engineer a delivery device (a Biodome) to monitor and control these factors (Figure 4). Control of the microenvironment through specific compounds delivered via an overlying Biodome will allow for directed differentiation of recruited cells into functional, innervated and vascularized soft tissue.
Figure 4: One example of a large animal Biodome prototype.
Agrawal, V., et al., Epimorphic regeneration approach to tissue replacement in adult mammals. Proc Natl Acad Sci U S A, Feb 2010. 107(8): p. 3351-3355.
Agrawal V, Tottey S, Johnson SA, Freund JM, Siu BF, Badylak SF. Recruitment of Progenitor Cells by an Extracellular Matrix Cryptic Peptide in a Mouse Model of Digit Amputation. Tissue Eng Part A. Vol. 17, No. 19-20, October 2011: 2435-2443.
Badylak, S. F., Valentin, J., Ravindra, A., McCabe, G., Stewart-Akers, A. Macrophage Phenotype as a Determinant of Biologic Scaffold Remodeling. Tissue Eng Part A, 2008 Nov; 14(11):1835-1842.
Brown BN, Londono R, Tottey S, Zhang L, Kukla KA, Wolf MT, Daly KA, Reing JE, Badylak SF. Macrophage phenotype as a predictor of constructive remodeling following the implantation of biologically derived surgical mesh materials. Acta Biomater, 2012 Mar;8(3):978-87.