Mammals fail to regenerate organs when wound contraction drives scar formation.

To understand why mammals generally do not regenerate injured organs, we considered the exceptional case of spontaneous skin regeneration in the early lamb fetus. Whereas during the early fetal stage skin wounds heal by regeneration, in the late fetal stage, and after birth, skin wounds close instead by scar formation. We review independent evidence that this switch in wound healing response coincides with the onset of wound contraction, which is also enabled during late fetal gestation.

Neural stem cell delivery via porous collagen scaffolds promotes neuronal differentiation and locomotion recovery in spinal cord injury.

Neural stem cell (NSC) grafts have demonstrated significant effects in animal models of spinal cord injury (SCI), yet their clinical translation remains challenging. Significant evidence suggests that the supporting matrix of NSC grafts has a crucial role in regulating NSC effects. Here we demonstrate that grafts based on porous collagen-based scaffolds (PCSs), similar to biomaterials utilized clinically in induced regeneration, can deliver and protect embryonic NSCs at SCI sites, leading to significant improvement in locomotion recovery in an experimental mouse SCI model, so that 12 weeks post-injury locomotion performance of implanted animals does not statistically differ from that of uninjured control animals.

Hesitant steps from the artificial skin to organ regeneration.

This is a historical account of the steps, both serendipitous and rational, that led my group of students and colleagues at MIT and Harvard Medical School to discover induced organ regeneration. Our research led to methods for growing back in adult mammals three heavily injured organs, skin, peripheral nerves and the conjunctiva. We conclude that regeneration in adults is induced by a modification of normal wound healing.

Regeneration mechanism for skin and peripheral nerves clarified at the organ and molecular scales.

This article is a review of current research on the mechanism of regeneration of skin and peripheral nerves based on use of collagen scaffolds, particularly the dermis regeneration template (DRT), which is widely used clinically. DRT modifies the normal wound healing process, converting it from wound closure by contraction and scar formation to closure by regeneration. DRT achieves this modification by blocking wound contraction, which spontaneously leads to cancellation of scar formation, a process secondary to contraction.

Regeneration of injured skin and peripheral nerves requires control of wound contraction, not scar formation.

We review the mounting evidence that regeneration is induced in wounds in skin and peripheral nerves by a simple modification of the wound healing process. Here, the process of induced regeneration is compared to the other two well-known processes by which wounds close, i.e.

Surface biology of collagen scaffold explains blocking of wound contraction and regeneration of skin and peripheral nerves.

We review the details of preparation and of the recently elucidated mechanism of biological (regenerative) activity of a collagen scaffold (dermis regeneration template, DRT) that has induced regeneration of skin and peripheral nerves (PN) in a variety of animal models and in the clinic. DRT is a 3D protein network with optimized pore size in the range 20-125 µm, degradation half-life 14 ± 7 d and ligand densities that exceed 200 µM α1β1 or α2β1 ligands. The pore has been optimized to allow migration of contractile cells (myofibroblasts, MFB) into the scaffold and to provide sufficient specific surface for cell-scaffold interaction; the degradation half-life provides the required time window for satisfactory binding interaction of MFB with the scaffold surface; and the ligand density supplies the appropriate ligands for specific binding of MFB on the scaffold surface.

In Situ Quantification of Surface Chemistry in Porous Collagen Biomaterials.

Cells inside a 3D matrix (such as tissue extracellular matrix or biomaterials) sense their insoluble environment through specific binding interactions between their adhesion receptors and ligands present on the matrix surface. Despite the critical role of the insoluble matrix in cell regulation, there exist no widely-applicable methods for quantifying the chemical stimuli provided by a matrix to cells. Here, we describe a general-purpose technique for quantifying in situ the density of ligands for specific cell adhesion receptors of interest on the surface of a 3D matrix.

Spectral-resolved multifocal multiphoton microscopy with multianode photomultiplier tubes.

Multiphoton excitation fluorescence microscopy is the preferred method for in vivo deep tissue imaging. Many biological applications demand both high imaging speed and the ability to resolve multiple fluorophores. One of the successful methods to improve imaging speed in a highly turbid specimen is multifocal multiphoton microscopy (MMM) based on use of multi-anode photomultiplier tubes (MAPMT).

Scar formation and lack of regeneration in adult and neonatal liver after stromal injury.

Known as a uniquely regenerative tissue, the liver shows a remarkable capacity to heal without scarring after many types of acute injury. In contrast, during chronic liver disease, the liver responds with fibrosis, which can progress to cirrhosis and ultimately liver failure. The cause of this shift from a nonfibrotic to a fibrotic response is unknown.

3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation.

Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane.

Emerging rules for inducing organ regeneration.

We review the available evidence for regeneration of adult organs of very diverse nature and examine the applicability of simple rules that can be used to summarize these treatments. In the field of regenerative medicine no widely accepted paradigm is currently available that can guide formulation of new theories on the mechanism of regeneration in adults and open new directions for improved regeneration outcomes. The four rules have emerged from multiyear quantitative studies with skin and peripheral nerve regeneration using scaffold libraries based on a simple, well-defined collagen scaffold.

Common features of optimal collagen scaffolds that disrupt wound contraction and enhance regeneration both in peripheral nerves and in skin.

The adult mammal responds to severe injury of most organs spontaneously by wound contraction and scar formation, rather than by regeneration. In severe skin wounds, the ability of porous collagen scaffolds to induce regeneration was found to correlate strongly with a reduction in wound contraction rate. Here, we present quantitative evidence of a similar positive relationship between the extent of disruption of tissue contraction and quality of peripheral nerve regeneration in transected rat peripheral nerves.

Template for skin regeneration.

Background: Advances in critical care allowed survival of large total body surface burn patients in the 1970s, spawning the development of artificial skin for burn victims. Lack of dermis resulted in severe scarring and contractures. The physicochemical properties that are critical to dermal regeneration have subsequently been described, and a dermal regeneration template has been developed.

An optical method to quantify the density of ligands for cell adhesion receptors in three-dimensional matrices.

The three-dimensional matrix that surrounds cells is an important insoluble regulator of cell phenotypes. Examples of such insoluble surfaces are the extracellular matrix (ECM), ECM analogues and synthetic polymeric biomaterials. Cell-matrix interactions are mediated by cell adhesion receptors that bind to chemical entities (adhesion ligands) on the surface of the matrix.

Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells.

Previous experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals.

Biologically active collagen-based scaffolds: advances in processing and characterization.

A small number of type I collagen-glycosaminoglycan scaffolds (collagen-GAG scaffolds; CGSs) have unusual biological activity consisting primarily in inducing partial regeneration of organs in the adult mammal. Two of these are currently in use in a variety of clinical settings. CGSs appear to induce regeneration by blocking the adult healing response, following trauma, consisting of wound contraction and scar formation.

Design of a multiphase osteochondral scaffold. II. Fabrication of a mineralized collagen-glycosaminoglycan scaffold.

This paper is the second in a series of papers describing the design and development of an osteochondral scaffold using collagen-glycosaminoglycan and calcium phosphate technologies engineered for the regenerative repair of articular cartilage defects. The previous paper described a technology (concurrent mapping) for systematic variation and control of the chemical composition of triple coprecipitated collagen, glycosaminoglycan, and calcium phosphate (CGCaP) nanocomposites without using titrants. This paper describes (1) fabricating porous, three-dimensional scaffolds from the CGCaP suspensions, (2) characterizing the microstructure and mechanical properties of such scaffolds, and (3) modifying the calcium phosphate mineral phase.

Design of a multiphase osteochondral scaffold. I. Control of chemical composition.

This is the first in a series of articles that describe the design and development of a family of osteochondral scaffolds based on collagen-glycosaminoglycan (collagen-GAG) and calcium phosphate technologies, engineered for the regenerative repair of defects in articular cartilage. The osteochondral scaffolds consist of two layers: a mineralized type I collagen-GAG scaffold designed to regenerate the underlying subchondral bone and a nonmineralized type II collagen-GAG scaffold designed to regenerate cartilage. The subsequent articles in this series describe the fabrication and properties of a mineralized scaffold as well as a two-layer (one mineralized, the other not) osteochondral scaffold for regeneration of the underlying bone and cartilage, respectively.

Design of a multiphase osteochondral scaffold III: Fabrication of layered scaffolds with continuous interfaces.

There is a need to improve current treatments for articular cartilage injuries. This article is the third in a series describing the design and development of an osteochondral scaffold based on collagen-glycosaminoglycan and calcium phosphate technologies for regenerative repair of articular cartilage defects. The previous articles in this series described methods for producing porous, three-dimensional mineralized collagen-GAG (CGCaP) scaffolds whose composition can be reproducibly varied to mimic the composition of subchondral bone, and pore microstructure and mineral phase can be modified.

Microarchitecture of three-dimensional scaffolds influences cell migration behavior via junction interactions.

Cell migration plays a critical role in a wide variety of physiological and pathological phenomena as well as in scaffold-based tissue engineering. Cell migration behavior is known to be governed by biochemical stimuli and cellular interactions. Biophysical processes associated with interactions between the cell and its surrounding extracellular matrix may also play a significant role in regulating migration.

Collagen-based matrices with axially oriented pores.

The aim of this work was the implementation of a simple technique for the production of cylindrical collagen-based scaffolds with axially oriented pore channels. Matrices with this particular porous structure have the potential to improve the regeneration of peripheral nerves and spinal cord by physically supporting and guiding the growth of neural structures across the site of injury. The regenerative potential may be further enhanced when the collagen scaffold is used as a delivery vehicle for exogenous cells and growth factors.

Standardized criterion to analyze and directly compare various materials and models for peripheral nerve regeneration.

Progress in understanding conditions for optimal peripheral nerve regeneration has been stunted due to lack of standardization of experimental conditions and assays. In this paper we review the large database that has been generated using the Lundborg nerve chamber model and compare various theories for their ability to explain the experimental data. Data were normalized based on systematic use of the critical axon elongation, the gap length at which the probability of axon reconnection between the stumps is just 50%.

Early fetal healing as a model for adult organ regeneration.

Evidence is provided pointing out certain basic similarities, though not an identity, between the mechanisms of early fetal regeneration and induced organ regeneration in adults. These similarities favor a model of induced organ regeneration in which biologically active scaffolds block wound contraction and scar formation.

Tissue engineering and developmental biology: going biomimetic.

This article contains the collective views expressed at the first session of the workshop "Tissue Engineering--The Next Generation," which was devoted to the interactions between developmental biology and tissue engineering. Donald Ingber discussed the chasms between developmental biology and tissue engineering from the perspective of a cell biologist who has had interest in tissue engineering since its early days. Van C.