Regenerative Capacity

In addition, the regenerative capacity of the olfactory epithelium raises the possibility that some of its cell types may prove useful for autologous transplantation therapies for damage to the central nervous system.

From: Reference Module in Biomedical Sciences , 2015

Self-assembled nanostructures for bone tissue engineering

Lei Yang , in Nanotechnology-Enhanced Orthopedic Materials, 2015

Abstract

The regenerative capacity of bone has been utilized to treat the bone fracture or defect below critical sizes for years. Based on this capacity and synergy of biomaterials and cell growth factors, a new bioengineering approach for bone restoration and regeneration, also known as bone tissue engineering, has been developed to treat large-size or complicated discontinuity problems in bone. With the assistance of nanotechnology, efficacy and potential of tissue engineering strategies for treating orthopedic problems have been substantially improved and the applicable scope of bone tissue engineering has been greatly enlarged. This chapter covers the fundamentals and examples of bone tissue engineering enabled by self-assembled nanostructures. Principles for self-assembly and fabrication methods of tissue engineering scaffolds are examined first. The rest of the chapter then elucidates the biomimetic nature and applications of self-assembled nanostructures for bone regeneration and restoration.

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Mending the Heart Through In Situ Cardiac Regeneration

J.C.M. Teo , ... N. Christoforou , in In Situ Tissue Regeneration, 2016

Intrinsic Regenerative Capacity of the Mammalian Heart

The intrinsic regenerative capacity of the mammalian heart is small when compared to other organs, suggesting that injury to the myocardium can have long-term deleterious effects [7]. A single major myocardial infarction incident can quickly lead to the elimination of up to 25% of the 2–4   billion cardiomyocytes that comprise the contractile machinery of the left ventricle [8]. Additionally, aging or long-term cardiac overburden as a result of chronic disease, such as hypertension, can also induce a significant loss of cardiomyocytes [9,10]. Thus in the absence of a significant induction of regeneration in the injured, ailing, or aging myocardium, the end result is heart failure and eventual death. Importantly, though, by understanding the small but definitive innate capacity of the heart to regenerate as well as the mechanisms that prevent it from doing so robustly, we can engineer tools that would ultimately allow us to otherwise induce a strong in situ cardiac regeneration, potentially allowing the complete functional recovery of the organ.

During development, both the human and rodent fetal hearts grow through proliferation of diploid mononucleated cardiomyocytes. Cardiomyocytes in human hearts continue to proliferate for the first months following birth, while in rodents, cardiomyocytes withdraw from the cell cycle within the first few days. In both species, this is followed by a significant increase in size of the cell cycle-withdrawn cardiomyocytes. The majority of rodent cardiomyocytes are binucleated with diploid nuclei as a result of a final nuclear division without undergoing cytokinesis [11]. Human cardiomyocytes, however, remain mononucleated and tetraploid, as the final nuclear division is not followed by cytokinesis [12]. This usually changes upon exposure to a pathophysiology, such as hypertension or cardiac overload due to myocardial infarction. Although the human cardiomyocytes remain mononucleated, they initiate DNA synthesis without undergoing division or proliferation leading to polyploid cells [12].

Unlike the mammalian heart, the zebrafish heart is capable of complete regeneration following surgical amputation. Cardiac regeneration in the zebrafish is due to the induction of proliferation of preexisting cardiomyocytes and not due to the generation of new cells from a population of progenitor cells [13]. The capacity of preexisting cardiomyocytes to proliferate following injury is almost completely absent in the mammalian rodent heart, and a recent study has revealed that only ∼0.008% of cardiomyocytes undergo division in the boundary region of the injured myocardium [14]. However, another possibility is that the rodent myocardium could regenerate due to the presence of a stem cell or progenitor cell population capable of differentiating into nascent cardiomyocytes. Studies have demonstrated that although such an effect does not take place during normal aging, following myocardial injury, a limited endogenous regenerative mechanism is indeed activated and is dependent on the presence of a progenitor cell population, rather than the induction of proliferation of preexisting cardiomyocytes [15]. Interestingly, a recent study has shown that the rodent heart can indeed completely regenerate following induction of injury similar to that performed in zebrafish, but this capacity is lost within the first 7   days following birth, suggesting that molecular events activated early in the mammalian life prevent future innate cardiac regeneration [16].

Human cardiac regeneration has been difficult to be confirmed definitively, due to the fact that macroscopically regeneration has not been observed, and at the cellular level, cardiomyocytes reinitiate DNA synthesis without undergoing cytokinesis following injury or overload. This translates into more or less the same number of cardiomyocytes but with increasing nuclear ploidy levels. When attempting to meticulously quantify the cardiomyocyte content in the human myocardium, it becomes apparent that the number of cardiomyocyte nuclei in the healthy heart is constant, and it only increases linearly following induction of hypertrophy, thus suggesting that human cardiac regeneration does occur at low levels due to pathological events [7,17,18]. A recent study elegantly attempted to accurately measure the amount of cardiomyocyte nuclei by taking advantage of the transient spike of atmospheric C14 concentrations, which occurred during the Cold War [19]. They determined that approximately 55% of human cardiomyocytes persist throughout the life of an individual, whereas 45% gets renewed.

Taken together, this evidence suggests that a baseline level of regeneration in the human heart does occur, although clearly this is insufficient to achieve full functional recovery to the organ due to the effects of aging, chronic disease, or injury. Moreover, it is not clear whether this is a result of proliferation of an existing population of cardiomyocytes or through the differentiation of a particular intrinsic stem cell or progenitor cell population. This, however, suggests that it is possible to engineer specific therapeutic tools that could either significantly enhance the existing baseline levels of human cardiac regeneration or inhibit the molecular mechanisms that prevent it from occurring more robustly with the ultimate goal of achieving complete organ regeneration and full functional recovery.

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Volume 2

Chiara Chiavelli , ... Graziella Pellegrini , in Encyclopedia of Tissue Engineering and Regenerative Medicine, 2019

Stem Cells and Self Repair

The long-term regenerative capacity of the corneal epithelium relies on the function of its stem cell pool. Under homeostatic conditions, corneal epithelial stem cells proceed to cell division producing mainly committed progenitors (transient amplifying cells), which in turn generate terminally differentiated cells. The cornea also includes a narrow transitional area between the bulbar conjunctiva and cornea, referred to as limbus. Limbal epithelium is composed of several layers of epithelial cells organized in well-developed rete ridges, known as the palisades of Vogt, populated by melanocytes, Langerhans's cells and corneal epithelial stem cells.

In 1986, the limbal area was first defined in a rabbit model as the location of corneal progenitors and putative stem cells. Afterward in humans, centripetal migration of cells from the limbal area was reported in patients with extensive ocular surface abrasions, leading to a later identification of cells endowed with long-term regenerative capacity, namely holoclones.

The formal demonstration that transplantation of corneal stem cells could restore the limbus and corneal integrity arose from the first conjunctival limbal autologous transplantation (CLAU) in total unilateral LSCD. This approach required the withdrawal of a large section of limbal-conjunctival tissue from healthy eyes.

Once the stem cell localization was defined, many studies have been focused on the identification of this population of cells both in vivo and in vitro. Due to evidence that TP63 transcription factor null mice displayed an impairment in all stratified epithelia development, this protein was investigated as a putative marker of corneal epithelial stem cells. Data collected from clonal analysis of limbal–corneal epithelial cells demonstrated that TP63 was highly expressed in long-term regenerating cells. Later, other markers were associated with human limbal stem cells (e.g., ABCG2, C/EBPδ, Bmi-1, N-cadherin, NGF/TrkA, Integrin α6, p75, Importin-13, CD38/157, ABCB5, WNT7A, K14), but so far TP63 is the only one associated with successful transplantation in clinical trials.

Unlike corneal epithelial cells, human corneal endothelial cells (HCECs) have a limited proliferative capacity in vivo. In fact, corneal endothelial cell loss is mainly counteracted by enlargement and migration of neighboring cells. HCEC quiescence is a result of strong contact inhibition and the presence of negative factors that prevent entry into the S-phase, such as TGF-β2. The existence of human corneal endothelial stem cells is yet to be proven. However, it has been hypothesized that putative endothelial stem cells can be located in the extreme periphery of mature corneal endothelium. These slow-cycling endothelial cells express embryonic cell markers, such as Oct-3/4 and Wnt-1, Pax-6 and Sox-2. This population might take part in endothelial regeneration and repair.

The stroma is populated by neural crest-derived mesenchymal cells, called keratocytes. In adult tissue, these cells display a dendritic morphology and are quiescent. Because of trauma or diseases, keratocytes undergo an activation process, becoming mitotically active and motile fibroblasts. This event leads to stroma remodeling and fibrotic extracellular matrix (ECM) deposition. This remodeling generates scar formation and contraction, inducing loss of corneal transparency. The later stages of these changes are characterized by α-smooth muscle actin fibroblast expression. Putative corneal stromal stem cells (CSSCs) were initially identified as a side population, located mostly in the anterior stroma, subjacent to the epithelial basement membrane, in the limbal region. These FACS-isolated cells, characterized by mesenchymal stem cells properties, were expanded clonally and they could reach up to 100 cumulative population doublings. Moreover, this population could be identified by the expression of specific markers, such as ABCG2, BMI-1 and PAX6. Experiments done in a mouse model with corneal opacity revealed that these CSSCs, isolated from human limbal stromal region, were able to restore corneal transparency. Some studies also investigated CD34 as a possible stromal stem cell marker, but it seemed to be a marker of keratocytes phenotype.

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Tissue Engineering of the Nervous System

Paul D. Dalton , ... Giles W. Plant , in Tissue Engineering (Second Edition), 2014

learning Objectives

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To understand the different regenerative capacities of the peripheral nervous system (PNS) and central nervous system (CNS), and identify the pros and cons of using different cell types for transplantation into the injured CNS

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To appreciate the critical gap length in peripheral nerves, and how therapies can increase this distance

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To gain a knowledge of different bioengineering strategies being used to promote peripheral nerve repair

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To understand the potential role of genetic modification of cells for use in biohybrid implants in the nervous system, and the limitations of animal models for human neurotrauma and neurodegenerative disease

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To recognize the different types of spinal cord injury (SCI) and how this affects potential treatment strategies

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To understand the importance of using biologically relevant peptide sequences in polymer scaffolds

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To appreciate the unique nature of the optic nerve as a model for CNS regeneration

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To appreciate a key challenge of astrocyte scarring to neuroprosthetic arrays

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Magnetic resonance imaging of ischemic heart disease

Ahmed Abdel Khalek Abdel Razek , ... Germeen Albair Ashmalla , in Cardiovascular and Coronary Artery Imaging, 2022

7.3.1 Infarct size and extent of transmural involvement

Myocardial tissue is characterized by low regenerative capacity with replacement of irreversibly damaged myocardium by nonfunctional fibrotic scar. Thus the amount of lost contractile tissue is strongly correlated with worse LV remodeling and patient outcome. LCE imaging proved to be a well-validated, accurate, and reproducible way to correctly measure the size of myocardial infarct no matter the age of infarct. It can detect small-sized infarcts that are not detected by ECG or other imaging modalities such as SPECT. Moreover, assessment of infarct transmurality is the second clue of infarct severity. Several studies stated that the more mural thickness involved by infarct the less inotropic reserve, less functional recovery of contractility, more severe residual adverse effects; severe post-infarct thinning of myocardial wall, ventricular wall aneurysm, and ventricular wall remodeling [18–22].

Early measurement of the infarct size is usually overestimated as it will be affected by the presence of edema and other cellular elements [21]. Progressive decrease of the infarct size occurs due to improvement of tissue edema and gradual shrinkage of scar tissue (by as much as 25% over a period of 4–8 weeks). Another factor is the increase in the remaining myocardial mass attributed to compensatory hypertrophy. So, the scar to normal myocardium ratio appears to become smaller by time. These changes are not well represented in calculations of LV total volume and mass [22].

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Three-dimensional (3D) printing based on controlled melt electrospinning in polymeric biomedical materials

Yong Liu , ... Seeram Ramakrishna , in Melt Electrospinning, 2019

8.1 Introduction

Biomaterial designs attempt to harness the regenerative capacity of the body, by merging principles of materials engineering and biological science, to repair damaged tissue in the body [1–3]. Over the last few decades, several multimodal biomimetic strategies have emerged to alleviate damaged tissue using a wide range of fabrication methods [4,5]. Of these, electrospinning has gained rapid recognition, for opening a new horizon in tissue engineering methodologies, owing to its simple yet precise methods to fabricate scaffolds with nano/macroscale topography [6–9]. Near-field electrospinning is a relatively new technique that utilizes an electrically charged polymer solution to deposit continuous fibrous meshes [10–12]. These meshes are porous, biocompatible, have a high surface area, and can be fabricated with advanced features such as drug elution of orientation from a range of polymers. Such scaffolds ultimately resemble the structure and size of the extracellular matrix natural tissues. This has made electrospinning an attractive strategy to produce surgical constructs for regenerative medicine. Although near-field electrospinning, as a concept, was proposed in 2006 [13], direct-write electrospinning research has experienced exponential growth in the past decade. This is attributed to the accuracy of the printing technique in recapitulating the micro/nanoscale composite structure required to meet the needs of individual patients [14–19].

In recent years, scientists have combined three-dimensional (3D) printing and electrospinning, and achieved significant milestones in biomedical design methods for tissue engineering. Many multidisciplinary research teams across the world have advanced the electrospun 3D printed precision and controllability, porosity, and mechanical properties of the scaffolds. Unlike nonbiological applications, 3D bioprinting with electrospinning involves posing significant technical challenges to cater for the sensitivity of living cells and the construction of functional tissues. Fabrication of such designs requires high geometric accuracy as well as the incorporation of complexities such as biomaterial choice, cell types, and bioactivity factors [20,21].

Three-dimensional printing in biomedical polymeric materials can be broadly split into four basic levels; (1) organic model manufacturing, (2) permanent implacable manufacturing, (3) indirect assembly of cells, and (4) direct cell assembly manufacturing [22–26]. At present, 3D printing has already found application in surgical analysis planning and manufacturing of prosthetic implants [27–31]. The augmentation of scaffold fabrication with 3D printing stands to deliver enormous sophistication and personalized solutions to tissue damage. Due to the high precision of the technology [15,32,33], electrospinning combined with 3D printing continues to evolve, in the hope of achieving specific outcomes in regenerative medicine through scaffold engineering, drug delivery, wound dressing, and enzyme immobilization [34–41]. With the rise in the aging population and increase in regenerative medical demands, the development of scalable and automated bioprinters will enormously impact the quality and affordability of medical care in the future.

In this review, we provide an in-depth understanding of 3D printer devices, with a focus on electrospinning underlining techniques, to produce fibers for biological morphology and application. Herein, we discuss the basic working principles and compare several additional features, such as electrostatic lens auxiliary electrodes, core–shells, the core structure of spray nozzles, and needle core induction receivers.

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Tissue engineering for female reproductive organs

Renata S. Magalhaes , ... Anthony Atala , in Principles of Tissue Engineering (Fifth Edition), 2020

Cell-seeded scaffolds for partial uterine repair

In an effort to enhance the regenerative capacity of tissue-engineered scaffolds, different cell sources combined with biomaterials have been explored. Atala et al. investigated the possibility of engineering autologous uterine tissue using a biodegradable polymer-based scaffold seeded with primary uterine cells [35,36]. Endometrium and myometrium cells were isolated and expanded ex vivo from rabbit uterine horns and seeded in a stepwise fashion onto the lumen and outer surface, respectively, of preconfigured semicircular scaffolds composed of polyglycolic acid (PGA)—coated poly-dl-lactide-co-glycolide (PLGA). The uterine construct was used to replace a subtotal excised horn in animals from which the cells derived. Six months after cell-seeded construct implantation, the neo-uterine tissue showed organized cellular and anatomical structures as well as expression of specific markers for epithelial, stroma, and smooth muscle cells. Functional studies are being conducted to assess the reproductive outcomes from the autologous bioengineered uteri.

Collagen scaffolds seeded with donor rat bone marrow–derived mesenchymal stem cells (BM-MSCs) have been studied in a murine model of severe uterine injury [37]. A suspension of labeled BM-MSCs (5×105 cells/cm2 scaffold) was seeded into collagen membranes (15×5   mm) and incubated for 1–3   hours before being engrafted in a partially removed uterine horn. Four weeks after surgery, BM-MSCs migrated to the basal layer of the graft, and the perigraft tissue had greater expression of bFGF, insulin-like growth factor 1, VEGF, and transforming growth factor-β1 than that in the collagen membrane-only group. Reproductive studies conducted 90 days after engraftment confirmed viable offspring in the BM-MSC/scaffold group suggesting that BM-MSCs may contribute to functional uterine tissue regeneration [37].

In another study, collagen scaffolds were loaded with endometrium-like cells derived from human embryonic stem cells (ESCs) and used as an implant in a rat uterine full-thickness injury model [38]. The results from in vivo studies indicated that although the cellularized collagen scaffold enhances uterine tissue regeneration, few human ESCs-derived cells were detected in the engrafted sites.

Other DC/recellularization techniques have been applied to create implantable grafts for uterine tissue regeneration. Miyazaki et al. [39] attempted to repopulate a whole rat decellularized uterine matrix injecting 5.1×107 neonatal uterine cells, 2.7×107 adult uterine cells, and 1.0×106 rat BM-MSCs in the uterine wall. After 3 days of incubation in a perfusion system, an endometrium-like tissue formation was observed, although cells were not evenly distributed within the matrix. When recellularized grafts (15×5   mm) were implanted in a partially excised rat uterine horn, uterine tissue ingrowth was noticed within the grafts, and pregnancy outcomes were reported to be nearly comparable to animals with an intact uterus. In another study, Hellström et al. [40] recellularized whole rat uterus matrices with donor primary heterogeneous uterine cells mixed with rat green fluorescent protein (GFP)-labeled MSCs. Grafts (20×5   mm) received multiple injections of a cell mix (one primary uterine cell per 150 GFP-MSCs) and were incubated for 72   hours. In vitro analysis of recellularized scaffolds revealed cell distribution limited to the matrices' external surface. Recellularized matrices (5×10   mm) were implanted in full-thickness excised rat uterine horns; and tissue ingrowth within the grafts was described at 6 weeks, mainly from host cells. Pregnancies in the remodeled uterine horn were reported, although embryo implantation did not occur directly in the grafts [40].

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Regenerative pharmacology and bladder regeneration

K.-E. ANDERSSON , G.J. CHRIST , in Biomaterials and Tissue Engineering in Urology, 2009

15.2 Endogenous bladder regeneration

The possibility to enhance the endogenous regenerative capacity of an organ such as the bladder, and to characterize pharmacologically and functionally the newly synthesized tissue offers a challenge for regenerative pharmacology. Studies to 'actively' enhance endogenous bladder regeneration still seem to be lacking, but this is not surprising given the paucity of information about the mechanistic basis of de novo bladder regeneration in vivo. Nonetheless, investigations using a 'passive' approach are emerging. For example, Frederiksen et al. (2004) studied the pharmacological and mechanical properties of newly developed detrusor muscle after subtotal cystectomy. They performed a partial, trigone-sparing cystectomy in female rats and explored, by pharmacological means, whether the regenerated detrusor had characteristics similar to the normal bladder base (supratrigonal segment), from which it regenerated, or to the normal bladder body (equatorial segment), which it replaced (see Fig. 15.2).

15.2. There is a regional variation in the bladder in response to electrical field stimulation and to drugs stimulating muscarinic, α-adrenergic and purinergic receptors.

Fifteen weeks after the operation, detrusor strips were cut from supratrigonal and equatorial segments (middle of the bladder body). Sham-operated rats served as controls. Responses to electrical field stimulation (EFS) were obtained in the absence of scopolamine (blocking muscarinic receptors), prazosin (blocking a1-adreneceptors) and α–β-methylene-ATP (desensitizing P2×1 receptors). Concentration–response curves were obtained for carbachol (stimulating muscarinic receptors), α–β-methylene-ATP (stimulating P2×1 receptors) and phenylephrine (stimulating α1-adrenoceptors).

The results revealed that the maximal contractile response to EFS was 60% of that to high-K+ solution (i.e. depolarization with KCl) in strips from both control and cystectomy bladders. Prazosin had no effect. Scopolamine decreased the maximal response of supratrigonal strips to 62% (controls or sham-operated) and 61% (operated or subtotal cystectomy) of that without blocker. For equatorial strips the decrease was to 81% (controls or sham-operated) and 58% (operated or subtotal cystectomy). Frequency–response relations were obtained during blockade with scopolamine, α–β-methylene-ATP and prazosin. Supratrigonal strips showed a pronounced additional inhibition up to 40 Hz. Equatorial strips from controls were completely inhibited at all frequencies. Equatorial strips from operated bladders were inhibited up to 20 Hz but not at 40 and 60 Hz. Computer analysis of the concentration-response curves, using the logistic equation, revealed that the carbachol EC50 values (concentration of carbachol that produced a response equivalent to 50% of the calculated maximal response) were similar in all groups. Moreover the calculated maximum response to phenylephrine (E max) was 10–20% of the high-K+ response. The authors concluded that there was a regional difference in pharmacological properties of normal detrusor, with a considerable contractile response to stimulation remaining in the supratrigonal muscle after simultaneous cholinergic, adrenergic and purinergic blockade. The new detrusor seemed functionally well innervated with no supersensitivity to muscarinic stimulation, and the newly formed bladder body had pharmacological properties specific for the supratrigonal segment from which it had developed.

In another initial report, urodynamic studies to evaluate bladder function in vivo, revealed that regenerated bladders were able to empty completely within 8 weeks after a subtotal cystectomy, albeit with slightly reduced volumes and with reduced maximal intravesical pressure (Burmeister et al., 2008; See Chapter 14 in this volume for more details). Taken together, the data suggest that this model is attractive for in vivo studies aiming to elucidate the mechanistic basis for de novo bladder regeneration in vivo. Such studies are an absolute prerequisite for developing pharmacological methods and technologies that can stimulate and enhance endogenous regeneration of bladder tissue by local or systemic administration of, for example, growth factors, cytokines, hormones, neurotransmitters and neuromodulators.

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Synthetic corneal implants

M.D.M. EVANS , D.F. SWEENEY , in Biomaterials and Regenerative Medicine in Ophthalmology, 2010

4.1.8 Corneal renewal and wound healing

Despite its avascular nature, the regenerative capacity of the corneal epithelium is considerable and occurs continuously in normal cornea to maintain the epithelium in good health ( Lemp and Mathers, 1991). The corneal epithelial replacement process was originally shown to take 5–7 days to complete in the rat cornea (Hanna and O'Brien, 1960), although more recent cell-labelling studies have shown it to take 2 weeks for complete turnover of the epithelium (Cenedella and Fleschner, 1990). Regeneration is primarily achieved by the migration of the epithelial cells from the periphery of the cornea inwards along the basement membrane and upwards to the superficial layers where they are continuously shed from the corneal surface into the tear film. This was proposed by Thoft and Friend (1983) in their X, Y, Z hypothesis of corneal epithelial cell replacement; where X represented proliferation in the basal cells, Y was the proliferation and migration of the limbal cells, and Z was the loss of epithelial cells from the ocular surface. Equilibrium in regular corneal epithelial cell replacement is maintained if X + Y = Z. The driving force for the centripetal migration of corneal epithelial cells is not fully understood, but is believed to involve desquamation of the central corneal epithelium in a preferential exfoliative process driven at least partly by the shearing force of the upper eyelid (Lavker et al., 1991; Lemp and Mathers, 1991; Mathers and Lemp, 1992). Davanger and Evensen (1971) first proposed that the regenerative capacity of the corneal epithelium resided in the papillary structure of the limbus at the corneal periphery, which is richly supplied with blood. Corneal epithelial stem cells were later identified in this location using monoclonal antibodies (Schermer et al., 1986; Zieske, 1992) and their slow-cycling nature while in the limbal microenvironment was demonstrated by thymidine labelling (Cotsarelis et al., 1989). Studies in skin (Watt, 1984; Jones and Watt, 1993) and then in cornea lead to the proposal that the pluripotent corneal epithelial stem cells from the limbus gave rise to semi-differentiated, multipotent 'transient amplifying cells' that migrated on to the cornea proper to divide and replenish the stratified epithelial layers of the central cornea as non-proliferative 'terminally differentiated cells' (Schermer et al., 1986).

Superficial wounding to the epithelium activates a rapid wound-healing response to quickly restore epithelial barrier function and normal vision. Epithelial cells around the wound periphery disassemble their hemidesmosomes and rearrange themselves, flattening and migrating inwards as a tissue front, which contracts in a 'purse-string' fashion to cover the defect (Buschke, 1949; Crosson et al., 1986; Gipson, 1989; Beuerman and Thompson, 1992; Dua et al., 1994). Once the wound is covered, the cells proliferate and stratify to restore normal epithelial thickness and hemidesmosomes reform in a wound-healing process that takes only days to complete (Hanna, 1966; Gipson, 1989; Dua et al., 1994). During this wound-healing phase, the undifferentiated epithelial stem cells located in the limbus and slightly differentiated 'transient amplifying cells' in the basal cell layer are stimulated to proliferate, providing additional cells to complete the restratification process (Dua and Azuara-Blanco, 2000). A direct linkage between differentiation status and proliferative capacity of corneal epithelial cells has been questioned in a study that showed that the daughter cells arising from cell division in the basal layer did not all differentiate synchronously to become wing cells, but rather, some remained in the basal layer with potential to undergo additional rounds of cell division (Beebe and Masters, 1996). Consistent with this are recent findings based on a human organotypic model where donut epithelial wounds were made to the central cornea, with dimensions 7 mm outer diameter and 3 mm inner diameter, and where the limbus was left intact or ablated. Data from the ablated limbus group showed that epithelial cells in the central corneal epithelium had the capacity to undergo sufficient cell division and migration to heal the epithelial wounds in the initial 12 hours post-surgery without recruiting cells from the limbus (Chang et al., 2008).

A substantially longer healing process of weeks to months is involved if the wound has penetrated both the epithelium and the stromal tissue (Stock et al., 1992; Jester et al., 1999). Wound repair in the corneal stroma is undertaken by the stromal keratocytes in a complex process modulated by soluble signalling molecules such as cytokines and growth factors such as platelet-derived growth factor (PDGF), keratocyte growth factor (KGF) and transforming growth factor-beta (TGF-β) that are produced by the injured epithelial cells above. It is not clear whether soluble signalling molecules derived from the epithelium penetrate the full thickness of the stroma or involve the interconnected keratocyte network in transmitting messages (Wilson et al., 2003). The response of the keratocytes to wounding is rapid and causes the keratocytes in the wounded stroma beneath to enter into programmed cell death known as 'apoptosis' (Wilson et al., 2003). Keratocytes in the stroma adjacent to the wound are activated to proliferate within hours of wounding and they transform into a fibroblastic phenotype and migrate into the affected area to repair damage (Fini, 1999; Jester et al., 1999; Wilson et al., 2003; West-Mays and Dwivedi, 2006). Repair fibroblasts may develop into a contractile phenotype known as myofibroblast during the wound-healing process and this is strongly mediated by the presence of TGF-β released from epithelial cells (Mohan et al., 2003). Activated fibroblasts and myofibroblasts synthesise and assemble new extracellular matrix, which has different components and properties to the normal uninjured stromal tissue (Funderburgh et al., 2003; Guo et al., 2007). Notable among these is hyaluronan, which is absent in the stroma of normal cornea but present in abundance in wounded corneas and those with chronic pathology and is regarded as a fibrotic matrix component (Guo et al., 2007).

Stromal-epithelial interactions are recognised to be a core part of the wound-healing processes that take place in response to corneal stromal injury (Melles et al., 1995). Significant to these interactions are the integrity of the epithelium and the exposure of keratocytes to epithelial-derived factors during wounding which determine whether corneal repair will be regenerative or fibrotic in nature (West-Mays and Dwivedi, 2006). Myofibroblasts and other cell types, such as bone marrow-derived cells, may be present in the stroma during the repair process depending on the nature and severity of the wound and their presence at the repair site causes haze (Dupps and Wilson, 2006). As the wound-healing response continues, stromal cells – such as keratocytes, fibroblasts, myofibroblasts and inflammatory cells – die by necrosis (Mohan et al., 2003). Damaged cells release pro-inflammatory chemokines which attract great numbers of bone-marrow-derived cells to clean up degenerative cells by engulfing debris into their cytoplasm in a process of phagocytosis (Dupps and Wilson, 2006). A study of myofibroblasts in tissues other than cornea (Dupps and Wilson, 2006) has suggested that myofibroblasts may originate in the bone marrow. These cells tend to be present in the stroma near the epithelium or sites of epithelial ingrowth into the stroma, implying that cytokines produced by epithelial cells are linked to their presence. Myofibroblasts are found where abnormalities of the stromal surface or regenerated basement membrane occur, as seen following refractive surgical procedures, such as surface laser ablation (Netto et al., 2006). Adult human corneal stromal wounds heal slowly and incompletely and may result in abnormalities such as scar tissue and reduplicated basement membrane (Melles et al., 1995; Dawson et al., 2008). These findings are consistent with the idea that the structural integrity of the epithelial basement membrane is significant in minimising the fibrotic response of the keratocytes and any subsequent scarring and loss of corneal clarity (West-Mays and Dwivedi, 2006).

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Extracellular Matrix as a Bioscaffold for Tissue Engineering

Brian M. Sicari , ... Stephen F. Badylak , in Tissue Engineering (Second Edition), 2014

5.5.1 Skeletal Muscle Reconstruction

Adult mammalian skeletal muscle tissue retains remarkable regenerative capacity following minor trauma such as exercise-induced injury. Specialized skeletal muscle progenitor cells called satellite cells are responsible for this regenerative plasticity. Following injury, signals from the injured skeletal muscle microenvironment stimulate resident satellite cells to proliferate and give rise to myoblasts, which go on to fuse and form contractile skeletal muscle myofibers. In the case of minor injury, these regenerated myofibers are able to recapitulate the injured skeletal muscle tissue.

When penetrating soft tissue injuries result in a massive loss of skeletal muscle tissue, the resulting defect is unable to be compensated for by the regenerative response of skeletal muscle. Such devastating trauma, referred to as volumetric muscle loss (VML), is commonplace on modern battlefields and results in significant cosmetic and functional deficits for wounded soldiers. Limited treatment options for VML, including autologous tissue transfer, are associated with complications such as donor site morbidity and failure of graft integration. The constructive tissue remodeling associated with surgically placed ECM bioscaffolds suggests a potential therapeutic application for VML treatment.

In vitro studies showed degradation products from ECM scaffold materials were chemotactic for myogenic skeletal muscle myoblasts and perivascular stem cells (Wolf et al., 2012; Crisan et al., 2008). In a mouse model, surgical placement of an ECM bioscaffold within sites of VML promoted the formation of islands of skeletal muscle cells after 56   days. Furthermore, in a preclinical canine model, an ECM bioscaffold remodeled into contractile skeletal muscle tissue after 6   months when placed within a VML defect affecting gastrocnemius skeletal muscle and associated Achilles tendon musculotendinous tissue (see State-of-the-Art Experiment textbox).

A recent clinical case study highlighted the ability of an acellular ECM scaffold to promote skeletal muscle constructive remodeling within a wounded soldier suffering from VML of the quadriceps femoris muscle (Mase et al., 2010). At 16   weeks postimplantation, the patient showed a 30% increase in muscle function that was concomitant with the presence of soft tissue consistent with skeletal muscle on computed tomography (CT) scan.

Furthermore, a clinical study is currently in progress, which evaluates the effect of ECM implantation at the site of VML in 80 patients. The outcome measures of this trial include mechanical strength and function, the cell populations involved in the process, quality of life in these patients, as well as to examine the cellular composition of the remodeled tissue. Preliminary results show the presence of multipotent perivascular cells, myosin heavy chain   +   skeletal muscle cells, neovascularization, and regenerating skeletal muscle tissue widely distributed throughout the remodeling ECM. Furthermore, at 24   weeks postoperatively these patients have exhibited at least a 25% increase in strength compared to presurgical values (Unpublished data). Taken together, these results demonstrate the potential clinical efficacy of ECM scaffolds in promoting the recruitment of perivascular progenitor cells and facilitating restoration of structure and function in volumetric skeletal muscle defects.

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