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. The crucial role of wound contraction in determining the wound healing outcome in adults has been demonstrated in three mammalian models of severe injury (excised guinea pig skin, transected rat sciatic nerve, excised rabbit conjunctival stroma) where grafting the injury with DRT, a contraction-blocking scaffold of highly-specific structure, altered significantly the wound healing outcome. While spontaneous healing resulted in scar formation in these animal models, DRT grafting significantly reduced the extent of wound contraction, prevented scar synthesis, and resulted in partial regeneration. These findings, as well as independent data from species that heal spontaneously via regeneration, point to a striking hypothesis: The process of regeneration lies dormant in mammals until appropriately activated by injury. In spontaneous wound healing of the late fetus and in adult mammals, wound contraction impedes such endogenous regeneration mechanisms. However, engineered treatments, such as DRT, that block wound contraction can cancel its effects and favor wound healing by regeneration instead of scar formation.

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. NSC-seeded PCS grafts can modulate key processes required to induce regeneration in SCI lesions including enhancing NSC neuronal differentiation and functional integration in vivo, enabling robust axonal elongation, and reducing astrogliosis. Our findings suggest that the efficacy and translational potential of emerging NSC-based SCI therapies could be enhanced by delivering NSC via scaffolds derived from well-characterized clinically proven PCS.

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. Contraction blocking by DRT is the result of a dramatic phenotype change in contractile cells (myofibroblasts, MFB) which follows specific binding of integrins α1ß1 and α2ß1 onto hexapeptide ligands, probably GFOGER and GLOGER, that are naturally present on the surface of collagen fibers in DRT. The methodology of organ regeneration based on use of DRT has been recently extended from traumatized skin to diseased skin. Successful extension of the method to other organs in which wounds heal by contraction is highly likely though not yet attempted. This regenerative paradigm is much more advanced both in basic mechanistic understanding and clinical use than methods based on tissue culture or stem cells. It is also largely free of risk and has shown decisively lower morbidity and lower cost than organ transplantation.

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., contraction and scar formation. Direct evidence supports the hypothesis that the mechanical force of contraction (planar in skin wounds, circumferential in nerve wounds) is the driver guiding the orientation of assemblies of myofibroblasts (MFB) and collagen fibers during scar formation in untreated wounds. We conclude that scar formation depends critically on wound contraction and is, therefore, a healing process secondary to contraction. Wound contraction and regeneration did not coincide during healing in a number of experimental models of spontaneous (untreated) regeneration described in the literature. Furthermore, in other studies in which an efficient contraction-blocker, a collagen scaffold named dermis regeneration template (DRT), and variants of it, were grafted on skin wounds or peripheral nerve wounds, regeneration was systematically observed in the absence of contraction. We conclude that contraction and regeneration are mutually antagonistic processes. A dramatic change in the phenotype of MFB was observed when the contraction-blocking scaffold DRT was used to treat wounds in skin and peripheral nerves. The phenotype change was directly observed as drastic reduction in MFB density, dispersion of MFB assemblies and loss of alignment of the long MFB axes. These observations were explained by the evidence of a surface-biological interaction of MFB with the scaffold, specifically involving binding of MFB integrins α1 ß1 and α2 ß1 to ligands GFOGER and GLOGER naturally present on the surface of the collagen scaffold. In summary, we show that regeneration of wounded skin and peripheral nerves in the adult mammal can be induced simply by appropriate control of wound contraction, rather than of scar formation.

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. This paper improves significantly the accuracy of the procedure introduced in a previous publication by detailed marker characterization, optimized staining, and improved data interpretation. The optimized methodology is utilized to quantify the ligands of integrins α 1 ß 1, α 2 ß 1 on two kinds of matched porous collagen scaffolds, which are shown to possess significantly different ligand density, and significantly different ability to induce peripheral nerve regeneration in vivo. Data support the hypothesis that cell adhesion regulates contractile cell phenotypes, recently shown to be inversely related to organ regeneration. The technique provides a standardized way to quantify the surface chemistry of 3D matrices, and a means for introducing matrix effects in quantitative biological models.

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). This approach improves imaging speed by using multiple foci for parallelized excitation without sacrificing signal to noise ratio (SNR) due to the scattering of emission photons. In this work, we demonstrate that the MAPMT based MMM can be extended with spectral resolved imaging capability. Instead of generating multiple excitation foci in a 2D grid pattern, a linear array of foci is generated. This leaves one axis of the 2D MAPMT available for spectral dispersion and detection. The spectral-resolved MMM can detect several emission signals simultaneously with high imaging speed optimized for high-throughput, high-contents applications. The new procedure is illustrated using imaging data from the kidney, peripheral nerve regeneration and dendritic morphological data from the brain.

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. These largely quantitative rules distinguish sharply between spontaneously regenerative and nonregenerative tissues, select the two reactants that are required for regeneration, recognize the essential modification of the wound healing process that must be realized prior to regeneration, and identify three structural features of scaffolds that are required for regenerative activity. The combined evidence points at certain requirements for the structure of a collagen scaffold with regenerative activity. An active scaffold emerges as a temporarily insoluble collagen surface, equipped with sufficient ligands for integrins of contractile cells, that inhibits wound contraction while also serving as a topographic template for new stroma synthesis. The four rules, based on studies with just two organs (skin and peripheral nerves), are now viewed in the context of ongoing studies using scaffolds based on decellularized matrices, which are mostly based on collagen. Decellularized matrices have been used during the past few years to regenerate, in whole or in part, the urethra, the abdominal wall, the Achilles tendon, the bladder, the trachea and other organs in several animal models and occasionally in humans. Although these acellular matrices are distinctly different from simple collagen scaffolds, and the methods used by the investigators are still evolving, the results obtained are shown to be broadly consistent with the predictions of the four rules. Future use or adaptations of these largely quantitative rules could account more satisfactorily for problems, such as imperfect function of regenerated organs, that are currently encountered by researchers. It could also further the explanation of the mechanism of regeneration at the cellular and molecular level.

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. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude.

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. Our observations suggest that porous collagen scaffolds enhance regeneration both in injured adult skin and peripheral nerves by disrupting the formation of a contractile cell capsule at the edges of the wound. Preliminary observations made with other injured organs support the hypothesis that capsules or clusters of contractile cells impose a universal mechanical barrier during wound healing which, if disrupted appropriately, enhances the quality of induced regeneration in a wider range of organs.

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. METHODS: The peer-reviewed literature regarding the basic science and clinical use of dermal regeneration template was reviewed, as was our practical experience in using dermal regeneration template. RESULTS: Dermal regeneration template has been effective in treating large areas of skin loss in burn victims and has been shown to have scar reductive and regenerative properties. Its use has been extended to reconstructive burns, including scar revisions and the treatment of burn scar contractures. It has also been useful in treating small areas of exposed bone, tendon, or joints and a variety of acute and chronic wounds. Meticulous wound bed preparation and close monitoring of the dermal regeneration template is critical to successful use. CONCLUSIONS: Dermal regeneration template provides a novel element to add to the reconstructive tools used today by plastic surgeons. Further development of these technologies may allow for improved regenerative capacity of these devices to allow optimal aesthetic and functional results of dermal reconstruction.

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. There are currently no established methods to obtain quantitative estimates of the density of adhesion ligands recognized by specific cell adhesion receptors. This article presents a new optical-based methodology for measuring ligands of adhesion receptors on three-dimensional matrices. The study also provides preliminary quantitative results for the density of adhesion ligands of integrins alpha(1)beta(1) and alpha(2)beta(1) on the surface of collagen-based scaffolds, similar to biomaterials that are used clinically to induce regeneration in injured skin and peripheral nerves. Preliminary estimates of the surface density of the ligands of these two major collagen-binding receptors are 5775 +/- 2064 ligands microm(-2) for ligands of alpha(1)beta(1) and 17 084 +/- 5353 ligands microm(-2) for ligands of alpha(2)beta(1). The proposed methodology can be used to quantify the surface chemistry of insoluble surfaces that possess biological activity, such as native tissue ECM and biomaterials, and therefore can be used in cell biology, biomaterials science and regenerative medical studies for quantitative description of a matrix and its effects on cells.

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. This article describes a method, "liquid-phase cosynthesis," that enables the production of porous, layered scaffolds that mimic the composition and structure of articular cartilage on one side, subchondral bone on the other side, and the continuous, gradual or "soft" interface between these tissues: the tidemark of articular joints. This design enables the layered scaffolds to be inserted into the subchondral bone at an osteochondral defect site without the need for sutures, glue, or screws, with a highly interconnected porous network throughout the entire osteochondral defect. Moreover, the differential moduli of the osseous and cartilaginous compartments enable these layered scaffolds to exhibit compressive deformation behavior that mimics the behavior observed in natural articular joints.

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. This article describes a technology through which the chemical composition-particularly the calcium phosphate mass fraction-of triple coprecipitated nanocomposites of collagen, glycosaminoglycan, and calcium phosphate can be accurately and reproducibly varied without the need for titrants or other additives. Here, we describe how the mineral:organic ratio can be altered over a range that includes that for articular cartilage (0 wt % mineral) and for bone (75 wt % mineral). This technology achieves the objective of mimicking the composition of two main tissue types found in articular joints, with particular emphasis on the osseous compartment of an osteochondral scaffold. Exclusion of titrants avoids the formation of potentially harmful contaminant phases during freeze-drying steps crucial for scaffold fabrication, ensuring that the potential for binding growth factors and drugs is maintained.

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. The methods build on the previously demonstrated ability to vary the composition of a CGCaP suspension (calcium phosphate mass fraction between 0 and 80 wt %) and enable the production of scaffolds whose pore architecture (mean pore size: 50-1000 microm), CaP phase chemistry (brushite, octacalcium phosphate, apatite) and crosslinking density (therefore mechanical properties and degradation rate) can be independently controlled. The scaffolds described in this paper combine the desirable biochemical properties and pore architecture of porous collagen-glycosaminoglycan scaffolds with the strength and direct bone-bonding properties of calcium phosphate biomaterials in a manner that can be tailored to meet the demands of a range of applications in orthopedics and regenerative medicine.

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. Although biophysical properties of two-dimensional substrates have been shown to significantly influence cell migration, elucidating factors governing migration in a three-dimensional environment is a relatively new avenue of research. Here, we investigate the effect of the three-dimensional microstructure, specifically the pore size and Young's modulus, of collagen-glycosaminoglycan scaffolds on the migratory behavior of individual mouse fibroblasts. We observe that the fibroblast migration, characterized by motile fraction as well as locomotion speed, decreases as scaffold pore size increases across a range from 90 to 150 mum. Directly testing the effects of varying strut Young's modulus on cell motility showed a biphasic relationship between cell speed and strut modulus and also indicated that mechanical factors were not responsible for the observed effect of scaffold pore size on cell motility. Instead, in-depth analysis of cell locomotion paths revealed that the distribution of junction points between scaffold struts strongly modulates motility. Strut junction interactions affect local directional persistence as well as cell speed at and away from the junctions, providing a new biophysical mechanism for the governance of cell motility by the extracellular microstructure.

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. The scaffold manufacturing technique described here is based on unidirectional freezing of a collagen suspension and subsequent freeze-drying, which produces nearly axially oriented pores. The mean pore size is dependent on both the concentration of collagen in suspension and the temperature of freezing. Environmental scanning electron microscopy and light microscopy were used to assess qualitatively and quantitatively the pore size and the pore orientation. In particular the definition of an orientation index (OI) was employed as a means to quantify the orientation of the pore channels inside the scaffolds.

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%. Use of this criterion has led to a rank-ordering of devices or treatments and has led, in turn, to conclusions about the conditions that facilitate regeneration. Experimental configurations that have maximized facilitation of peripheral nerve regeneration are those in which the tube wall comprised degradable polymers, including collagen and certain synthetic biodegradable polymers, and was cell-permeable rather than protein-permeable. Tube fillings that showed very high regenerative activity were suspensions of Schwann cells, a solution either of acidic or basic fibroblast growth factor, insoluble ECM substrates rather than solutions or gels, polyamide filaments oriented along the tube axis and highly porous, insoluble analogs of the ECM with specific structure and controlled degradation rate. It is suggested that the data are best explained by postulating that the quality of regeneration depends on two critical processes. The first is compression of stumps and regenerating nerve by a thick myofibroblast layer that surrounds these tissues and blocks synthesis of a nerve of large diameter (pressure cuff theory). The second is synthesis of linear columns of Schwann cells that serve as tracks for axon elongation (basement membrane microtube theory). It is concluded that experimental configurations that show high regenerative activity suppress the first process while facilitating the second.

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.

The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering.

The permeability of scaffolds and other three-dimensional constructs used for tissue engineering applications is important as it controls the diffusion of nutrients in and waste out of the scaffold as well as influencing the pressure fields within the construct. The objective of this study was to characterize the permeability/fluid mobility of collagen-GAG scaffolds as a function of pore size and compressive strain using both experimental and mathematical modeling techniques. Scaffolds containing four distinct mean pore sizes (151, 121, 110, 96 microns) were fabricated using a freeze-drying process. An experimental device was constructed to measure the permeability of the scaffold variants at different levels of compressive strain (0, 14, 29 and 40% while a low-density open-cell foam cellular solids model utilizing a tetrakaidecahedral unit cell was used to accurately model the permeability of each scaffold variant at all level of applied strain. The results of both the experimental and the mathematical analysis revealed that scaffold permeability increases with increasing pore size and decreases with increasing compressive strain. The excellent comparison between experimentally measured and predicted scaffold permeability suggests that cellular solids modelling techniques can be utilized to predict scaffold permeability under a variety of physiological loading conditions as well as to predict the permeability of future scaffolds with a wide variety of pore microstructures.