PI Stephen Badylak
Title Control of the Microenvironmental Niche to Promote Epimorphic Regeneration in Amputated Digits

Description Background: The proposed studies address one of the most pressing needs of the wounded soldier population: the replacement of lost digits and limbs. These studies challenge a fundamental tenet of the mammalian response to tissue injury; specifically, the concept that regeneration of complex tissues following severe injury is not possible in adult mammals. We hypothesize that such regeneration is indeed possible and, to support this hypothesis, we have successfully recruited endogenous stem cells to the injury site thru the use of chemotactic matricryptic peptides derived from extracellular matrix (ECM). We call this cell mass a “multipotential cell cluster” and it is similar, but not identical, to the true blastema of regenerating species such as the newt. We now seek support to identify the genetic signature, the appropriate microenvironmental niche, and inductive molecular signals required to stimulate functional tissue formation from the multipotential cell cluster.

Concept: We can effectively recruit abundant endogenous multipotential cells to the site of digit amputation in our well established mouse model. Proposed work would include isolation of individual cells that have been dissociated from the MCC and characterization of the transcriptome. The number of cells will be very low and will require cell sorting technology that our team has recently developed. Characterization of the transcriptome will be conducted by the J. Thomson/R. Stewart group at Madison, Wisconsin. Realization of the regenerative potential of the multipotential cell cluster will require control of the microenvironmental niche through development of a “biodome”. This work will be conducted by the team of Susan Braunhut in Boston, Massachusetts. The critical components of the proposed work include: 1) the mouse model and creation of the MCC, 2) MCC isolation and cell sorting, 3) characterization of the genomic profile and transcriptome, and 4) development of a biodome. This multidisciplinary project can only be accomplished through the combined efforts of multiple investigators and multiple novel technologies. The problem is challenging but the potential payoff is huge.

Objective/Hypothesis: To induce functional tissue formation in adult mammals by controlling the microenvironmental niche into which multipotential stem cells are recruited.

Specific Aims: 1) To identify the genetic signature that characterizes constructive tissue remodeling as opposed to scar tissue formation. 2) To produce a prototype biodome that can locally manipulate and control factors such as hydration state, pH, oxygen tension, electrical potential, and nutrient composition at the site of a multipotential cell cluster in a mouse model of digit amputation. 3) To form bone and functional contractile tissue at the site of digit amputation in a mouse model. 4) To develop and document the formation of a nerve and vascular plexus into the regenerated digit tissue.

Study Design: This two year project will utilize the mid-second Phalanx digit amputation model in C57Bl/6 mice. The work will be a progressive investigation of variables in the biodome that effect functional tissue reconstruction. In year one, in addition to developing the prototype biodome, we will evaluate oxygen tension and nutrient composition of the biodome fluid as variables that effect the cell transcriptome and functional tissue reconstruction. During year two, we will utilize the second version of the biodome device in which electrical potential, pH, and replacement of the biodome fluid can be investigated in systematic fashion. In addition, in year two, the analytic transcriptome comparison of the multipotential cell cluster (MCC) that develops in the C57Bl/6 mice will be compared against a carrier treated mouse control group and the transcriptome data that has been developed in separately funded work using the red spotted newt. This comparative transcriptome analysis will provide invaluable information regarding the efficacy of controlling cell phenotype and functional tissue reconstruction.

Continued iterative versions of the biodome will be utilized over the two years of this work. As opposed to what would be possible in one year, which is development of a single prototype, the second year will provide the opportunity to add controllable variables and develop a more user friendly version of the prototype. In addition, the shared number of biodomes developed and the size of the animal studies will increase dramatically thus adding to the study output.

Relevance: Loss of limbs and digits is one of the most serious lifelong consequences of the injured soldier population. In addition, the civilian population would benefit from the development of strategies to stimulate tissue regeneration as opposed to scar tissue formation.

Source ARM 3

Term 9/30/09 – 09/29/11

Amount: DC: $374,903 (for 2 years)
IDC: $52,226
TC: $427,129