Utility of extracellular matrix powders in tissue engineering

ABSTRACT

Extracellular matrix (ECM) materials have had remarkable success as scaffolds in tissue engineering (TE) and as therapies for tissue injury whereby the ECM microenvironment promotes constructive remodeling and tissue regeneration. ECM powder and solubilized derivatives thereof have novel applications in TE and RM afforded by the capacity of these constructs to be dynamically modulated. The powder form allows for effective incorporation and penetration of reagents; hence, ECM powder is an efficacious platform for 3D cell culture and vehicle for small molecule delivery. ECM powder offers minimally invasive therapy for tissue injury and successfully treatment for wounds refractory to first-line therapies. Comminution of ECM and fabrication of powder-derived constructs, however, may compromise the biological integrity of the ECM. The current lack of optimized fabrication protocols prevents a more extensive and effective clinical application of ECM powders. Further study on methods of ECM powder fabrication and modification is needed.

INTRODUCTION

Extracellular matrix (ECM) has been widely used in tissue engineering (TE) as a platform for de novo tissue formation and in regenerative medicine (RM) as a material to treat tissue injury by preventing scar formation and promoting constructive remodeling¹ The current uses of ECM powder are examined with focus on the novel applications afforded by its particulate form. Means by which powdering improves efficacy of ECM treatment in specific tissues, challenges inherent to powder fabrication, and factors preventing further application of ECM powder are analyzed.

The ECM refers to a network of fibrous and gel-like materials produced by resident cells. It exists in a state of dynamic reciprocity providing signals and cues to cells which in turn modulate the composition and organization of the ECM.² Specifically, the ECM creates a microenvironment that promotes constructive tissue remodeling, modulates gene expression and facilitates cell adhesion, migration, differentiation, and proliferation. ³

The interactions between cells and ECM motifs are highly important in determining cell fate and maintaining cellular phenotyp.⁴ In order to induce formation of the correct tissue at a specific site, the structure, composition, and source of ECM used must be considere.⁵ The macrostructure of ECM can be modified to suit the intended application. ECM may retain the conformation of the tissue it formerly surrounded or can be processed to form novel constructs.

ECM material that retains its original conformation, such as decellularized organs and sheets, provides the benefit of an intact vasculature, a precise mold of its derivative tissue, and preserved mechanical strengt.⁶ Although these forms of ECM undergo fewer processing steps, their clinical utility is somewhat limited because their structure prevents conformational adaptation.

ECM can be comminuted to yield powder which can be used in its particulate form or be enzymatically solubilized for use as a liquid or gel. ECM powder and powder-derived constructs (ECM liquid suspensions and gels) can fill areas of volumetric injury and conform to the contours of native tissues. The adaptable conformation of powder and its solubilized form allow for minimally invasive implantation such as injection. Unlike constructs with a fixed conformation which exhibit a proclivity to contract, particulate and solubilized ECM maintain the shape that is adopted.⁷

The adaptable form of powder is not only spatially advantageous, but also allows for efficient and homogenous conjugation to additional molecule. Powder can therefore act as a vehicle for delivery of cells, growth factors, and drugs. The dynamic properties of ECM powder render it effective in 3D bioprinting and treatment of a wide array of tissue injury. Although ECM powder and derived constructs seemingly offer infinite forms of application in TE and RM, challenges in fabrication and protocol optimization prevent a more extensive use.

Preparation of ECM powder involves extensive processing that potentially alters the biological integrity of the ECM. Steps in processing can affect the content, mechanical strength, ultrastructure and microstructure of the ECM.⁸ Preparation of powder ECM typically involves separation of the ECM from unwanted tissues, decellularization, freezing, lyophilization, pulverization, and milling. (Figure. 1) Conceivably, ECM components are disrupted at each step in fabrication and process may, in turn, alter the host response upon in vivo implantation.⁹

Despite widespread use of ECM powder, standard guidelines and sound methods for powder fabrication and treatment have not been defined. Fabrication and subsequent modification pose challenges to expanded application of ECM powders. This review examines the current uses of ECM powders and powder-derived constructs and challenges posed to expanding their applications in tissue engineering and regenerative medicine.

FIGURE 1.

Schematic represents the process of ECM comminution and illustrates the steps involved in powder fabrication. In this example, ECM is obtained by organ decellularization represented by change in color of the liver depicted above. These processes are not specific to a hepatic model.

FABRICATION OF POWDER AND POWDER-DERIVED CONSTRUCTS

The fabrication process poses challenges to the effective use of ECM powder and powder-derived constructs. In general, preparation of ECM powder involves separation of the ECM from unwanted tissues, decellularization, freezing, lyophilization, pulverization, and milling (Figure. 1). The biological integrity of ECM can potentially be compromised at each step.^{8, 9} Moreover, comminution and subsequent solubilization can alter in vivo response after implantation. Pulverization of porcine urinary bladder (pUBM) has yielded a product with preserved ultrastructure and 3D characteristics of ECM, but this has not been consistently reproducible in all models.¹⁰ Despite reports of constructs exhibiting desired bioactivity, tissue specific parameters for optimal powder fabrication have not been determined. To optimize the utility ECM powder considerations should be given, but not limited to: the amount and concentration of powder used in constructs, particle size and morphology, powder solubilization, and ECM crosslinking. These methods and protocols must also be reproducible if these constructs are to be used in a clinical setting.

Powder concentration

The amount of ECM powder and concentration of said powder in liquids or gels influences the mechanical and physical properties of these constructs. The concentration of ECM powder in subsequently solubilized constructs affects porosity, compressive modulus, gelation threshold, and rheological properties. The amount of ECM material implanted can also affect the process of tissue remodeling. Notably, excessive ECM deposition in vivo can produce tissue fibrosis.¹¹ Therefore, it is important to determine the appropriate quantity of ECM needed for regeneration and repair of specific tissues.

Scaffolds with a low ECM concentration are highly porous, but exhibit the tendency to contract. Increasing the concentration of ECM in a scaffold decreases the porosity but prevents significant scaffold contraction.¹² Decreasing ECM concentration improves cellular adhesion and proliferation; hence there is a tradeoff between fabricating a scaffold that has greater biological activity and one that effectively fills areas of tissue deficit. ¹³

To create an effective scaffold, the concentration of ECM powder must therefore be adjusted to balance the poor cell-scaffold interactions of highly concentrated ECM scaffolds against the issue of contraction in scaffolds with a lower ECM concentration that have greater cell-scaffold interactions.

ECM concentration also influences gelation of solubilized powder and subsequent rheological properties of these gels. Hydrogels with higher concentrations of ECM exhibit a rigid structure with defined edges, whereas hydrogels with lower concentrations of ECM were more pliable and had rounder edges. Significantly, the effect of concentration on pore size of hydrogel was dependent upon the type of ECM used. The concentration of urinary bladder matrix (UBM) did not have any effect on hydrogel porosity, whereas increasing concentration of dermal ECM decreased hydrogel pore size. Therefore, the amount and concentration of ECM used is dependent upon the type of ECM used.¹⁴

Increasing the concentration of ECM increases the viscosity of powder suspensions causing gelation. The rheological properties of the resulting gels are highly significant clinically in that highly concentrated ECM hydrogels may not pass through needle lumen, preventing therapeutic injection.¹⁴ The properties of hydrogels have not been well characterized, but there is sufficient evidence to suggest that the concentration of ECM powder used in their fabrication has significant bearing on these properties. ¹⁴

Particle conformation

Despite biological composition, ECM powder can induce different changes in vivo depending on particle conformation. Specifically, size and morphology of ECM particles can influence cell proliferation, differentiation, tissue development.^13,15,16 ECM powder has a wide distribution in particle size that is difficult to control.¹⁷ During powder preparation, residual moisture can induce clumping and agglomeration of powder can occur resulting in particles with numerous sizes. Separating agglomerations is difficult and clumps can be trapped in the milling process.¹⁸ Although particles with diverse shape and size are satisfactory substrates for cellular adhesion and growth,¹⁹ particles with uniform morphology allow for more control of tissue development in vivo. For example, use of powder with diverse particle sizes in skin TE diversity can manifest meshes with gaps which result in non-contiguous tissue formation that is cosmetically unaesthetic.¹⁷ Further study is needed to determine whether bioengineering of certain tissues may require specific particle sizes.

Sterilization

ECM biomaterials should be terminally sterilized before clinical use. Sterilization should effectively sanitize ECM by minimally destructive means. Many sterilization techniques and agents are used, including, but not limited to: gamma irradiation, electron-beam (e-beam), glutaraldehyde, ethylene oxide, peracetic acid (PAA.⁶ These methods have been assessed for their effect on the mechanical and biological integrity of ECM scaffolds, but have not been extensively studied in the context of ECM powder.

In general, ECM sterilization presents a challenge to manufactures: high temperatures denature matrix proteins,²⁰ irradiation can degrade growth factors within in the matrix,²¹ and soluble reagents may not effectively penetrate the matrix.²²

Specifically, sterilization of ECM powder requires further consideration because the effect of sterilization depends in part on whether ECM is hydrated or lyophilize.²³ For example, sterilization of lyophilized ECM with ethylene oxide and e-beam altered mechanical properties and enzymatic digestion resistance of the ECM. Sterilization by the same means did not alter properties of hydrated ECM.²⁴

Forms sterilization like PAA and ethanol that have limited effects on matrix properties, however, are not appropriate for powder. These aqueous forms of sterilization can alter particle size and morphology because powder may be solubilized or clum.²⁵ Further study is needed to determine the best means of sterilizing ECM particles for clinical application of ECM powder to be expanded.

Solubilization

Powder is often solubilized enzymatically to make homogenous liquids or gels. Enzymatic digestion requires an acidic pH; therefore, solubilization commonly uses in hydrochloric acid or acetic acid. For in vivo use, acidic solubilized powder must be neutralized to physiological pH. If neutralization is not successful, the ECM will provide an acidic microenvironment that hinders vascularization and induces inflammation, granulation, and fibrosis.²⁶

If enzymatic digestion was not sufficient or complete, particles may precipitate out of solution and the construct would have a heterogenous composition.¹⁸

Crosslinking and conjugation

Powder derived liquids and gels lack mechanical strength; therefore, ECM protein crosslinking is often used to strengthen their structure and inhibit degradation. Crosslinking is successfully strengthens hydrogels, but linker chemicals elicit a foreign body response in vivo.²⁷

Additionally, crosslinking can be used to conjugate additional molecules (e.g., drugs, fluorescent markers) to the matrix. Carbodiimide crosslinkers such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are often used to tether molecules to ECM collagen. EDC/NHS reaction with collagen, however, can leave toxic residues in the ECM.²⁸

APPLICATIONS IN TISSUE ENGINEERING (TE)

ECM powder as a vehicle

Pulverized ECM offer a unique modality for the delivery of biologic products including growth factors and cells. Additionally, comminuted forms of ECM can be conjugated with pharmacologic agents to achieve more complex drug release profiles with greater tunability.

Cell delivery and cell culture

ECM powder and subsequently solubilized powder can both act as vehicles for 3D cell culture and minimally invasive cellular delivery to specific sites. 3D cell culture has been previously achieved in sponges, spheroids, hydrogels, and decellularized organs, but these platforms do not allow for uniform cell distribution or optimal nutrient diffusion.

Powdered ECM processed into cell-sized microparticles, however, would potentially allow cells to incorporate homogenously with and uniformly attach to the particulate scaffold yielding an improved 3D culture model. Given the large surface area to volume ratio of ECM particles, cellular attachment to ECM would be maximized. Unlike existing 3D culture models, the properties of an ECM particle mixture, such as particle size and concentration, could be modulated to regulate cell-cell and cell-matrix interactions.

Fabrication of ECM microparticles has not yet been achieved. Collagen, the most abundant protein of ECM, has been successfully comminuted to cell-sized microparticles which were able to form spheroids with primary rat hepatocytes.²⁹ Basolateral cell-matrix and apical cell-cell interactions of multicellular spheroids induced cells to align radially and compactly in a single row fashion. The orientation of collagen-hepatocyte spheroids highlighted the potential of ECM microparticles to create 3D constructs with a complex microstructure. Further research on the effect comminution to a microparticle scale on the biological integrity of ECM is required. Formation of viable hepatocyte spheroids using collagen microparticles, however, suggests future potential of ECM microparticles.

Powdered ECM consisting of larger particles has already been used as cell carriers. Cells can be seeded directly on to powder¹⁹ or else suspended in a solution of solubilized powder. A cell-ECM suspension is particularly advantageous because it allows cells to be delivered via injection. Notably, grafts from injected solubilized ECM powder laden with human adipose-derived stem cells were superior to autologous adipose transplants which decreased in volume and size over time. Poor results from autologous grafts, which also contain native ECM, suggest that the use of solubilized particular ECM as a cell carrier conveys certain benefits that uncomminuted ECM may not.³⁰

Cell culture in solubilized ECM powder may also be advantageous in promoting differentiation of progenitor cells. Study on cardiac muscle TE, demonstrated that induced pluripotent stem cells (iPSCs) can differentiate into mature cardiomyocytes when grown in solubilized cardiac ECM powder. iPSCs usually have the proclivity to differentiate into cardiomyocytes with a fetal phenotype. Deviation from the characteristic behavior of these cells indicates that solubilized ECM powder may provide a microenvironment that induces progenitor cells to terminally differentiate.³¹ Moreover, cells coated in solubilized ECM powdered derived from their native environment have exhibited increased levels of proliferation and differentiation as well as maintenance of the specific cell phenotype.³²

As previously mentioned, ECM powder creates a 3D culture system in which cell-cell and cell-matrix interactions result in the unique assembly of cellular aggregates, such as spheroids. The ability of ECM powder to induce formation of cellular aggregates is significant because aggregates have been noted to function as a tissue unit. In a study on articular cartilage repair, mesenchymal stem cells incorporated with cartilage derived ECM powder formed functional “micro-cartilage” aggregates which induced hyaline like articular cartilage formation from of native stem cells and cells seeded on ECM particles. Significantly, ECM powder in this experiment provided a particulate carrier environment that could maintain the phenotype of chondrocytes which has the tendency to be lost over time. These aggregates were able to be delivered arthroscopically to sites of injury allowing for a one step, minimally invasive approach to cartilage repair.³³

Drug delivery

Successful pharmaceutical treatment requires effective drug delivery. Polymeric scaffolds, have already shown potential as drug carriers.³⁴ ECM powder, however, provides a more tunable and precise method of drug delivery. Powder can be homogenously conjugated with drugs to target specific sites in the body and to achieve more complex drug release profiles.

The bioavailability of drugs can be easily modulated by adjusting the concentration of ECM powder in the drug-ECM complex. Use of ECM powder in this manner has provided increased efficacy in controlled and sustained release of drugs.³⁵

Drug conjugation to powder has been used to create 3D printed drugs, such as levetiracetam.³⁶ 3DP allows for control of spatial drug distribution and accurate drug dispense at low volumes, thereby reducing waste in manufacturing drugs and creating a highly tunable release profile.^{37, 38} ECM powders have not been used in drug 3DP, but their separate uses in drug delivery and 3DP indicates potential future application in drug 3DP.³⁹

Novel use of a particulate system in a recent study on the treatment of non-small cell lung cancer identifies a drug delivery technique in which ECM powders may be applied. Nanoparticles and microparticles of chitosan encapsulated drug conjugated polyvinylpyrrolidone/maltodextrin were used as an inhalable treatment. Drug inhalation is non-invasive and specifically targets the lungs, preventing systemic adverse effects.⁴⁰ ECM particles, which have been used in repair of lung tissue,⁴¹ may potentially be used in as an inhalable drug carrier but with increased efficacy owing to the bioactivity of ECM.

Growth factor delivery

ECM acts as a reservoir for cytokines and signaling molecules. As remodeling occurs, these molecules can be released. Given these properties, it follows that ECM can be used to deliver exogenous growth factors (GF) to areas in the body. ECM powders are particularly effective in delivery because they are easily modified and can be solubilized for injection to specific parts of the body.

Powder has been used to deliver growth factors to areas that are difficult to access directly, such as the liver. Previously growth factor delivery to hepatic parenchyma was achieved solely through vasculature. Recent study demonstrated that liver-derived ECM powder could carry GFs for their direct delivery to hepatic parenchyma to induce hepatocyte proliferation and liver regeneration. This is highly significant because liver regeneration occurs via adult hepatocyte expansion which is dependent upon GFs and ECM.⁴²

Moreover, the powdered ECM has a high capacity to carry GFs and have good release profiles. An experiment on chondrogenesis using solubilized powdered ECM, increasing amounts of TGF-β3 were successfully loaded onto ECM which exhibited a release profile of GF that supported chondrogenesis well.^{13, 43}

Powder conjugation

ECM powder is easily and homogenously modified. It can act as a conjugate in which it augments or “ornaments” synthetic polymers and surgical materials to mitigate an inflammatory response. Alternatively, it can serve as a substrate to which molecules are conjugated.

Molecular labelling

ECM powder serves as an excellent substrate for bioconjugate reactions because it allows for sufficient penetration of chemical reagents and homogenous mixing.⁴⁴ Fluorescent labelling of ECM powder has been achieved by bioconjugate N-hydroxysuccinimide (NHS) reaction between fluorescent Cy3 molecules and matrix collagen. Significantly, fluorescently labelled solubilized powder allowed for real-time monitoring of scaffold degradation by minimally invasive injection which prevented destruction of samples.

ECM ornamentation

ECM powder has been successfully mixed with synthetic polymers to fabricate more biocompatible materials for use in TE and surgical wound closure. Specifically, dermal powder solubilized to make a hydrogel has been used to coat polypropylene surgical meshes. Polypropylene is non-degradable polymer which may elicit a foreign body response and significant inflammation. ECM ornamentation of the synthetic meshes, however, mitigates inflammation and subsequent fibrosis and scar tissue deposition. Modification by ECM hydrogel, which lacks mechanical strength, did not compromise the mechanical properties of surgical meshes. ECM ornamented surgical meshes were found to be effective in supporting weight bearing loads in the repair of ventral hernias, inguinal hernias and pelvic organ prolapse.^{45, 46} Polyurethane cardiac patches were coated with solubilized cardiac ECM powder. ECM prevented the progression of maladaptive remodeling after cardiac infarct and ultimately decreased left ventricular compliance, promoted angiogenesis, and prevented left ventricular dilatation, scarring, and thinning.⁴⁷

ECM has also been used to coat synthetic polymer scaffolds. Solubilized small intestine submucosa (SIS) ECM powder was used to coat polylactic-co-glycolic acid (PLGA) for use in nucleus pulposus regeneration of intervertebral discs. PLGA, which yields an acidic degradation product, exhibited increased biocompatibility when augmented by SIS-ECM. There was more cellular adhesion to the composite SIS-ECM-PLGA scaffolds than to PLGA alone.⁴⁸ Adding bone powder ECM to chitosan also increased bone regeneration and filled the area of bone defect better than a chitosan scaffold alone.⁴⁹ ECM, which can self-assemble, has been mixed in the powdered form with inorganic materials that do not self-assemble. This has been particularly useful in bone microfiber formation which usually uses inorganic apatite matrices that do not self-assemble.⁵⁰

3D PRINTING (3DP)

3DP is a bio-fabrication technique in which layer-by-layer assembly creates a complex 3D structure.⁵¹ Specifically, powder based 3DP has been used to make structurally complex scaffolds for application in TE.³⁸ Powder is “printed” following the 2D template of each layer and then sprayed with a liquid binder to solidify the powder.

3DP powder substrates are commonly synthetic polymers which have limited biological activity and poor biocompatibility when compared to their naturally occurring counterparts. Although 3DP with synthetic polymers successfully yields complex 3D structures, the use of ECM powder increases biological activity of the construct.⁵²

ECM powder can be solubilized and laden with cells to create “bio-ink” for use in 3DP. Bio-ink allows cells to be printed by preventing cellular damaging during the printing process. The viscosity of the solubilized ECM protects from shear stress induced apoptosis.⁵³

Bio-ink, like powder, must be stabilized layer-by-layer as it is printed. Solidification of bio-ink is vital to stabilizing the 2D printed pattern and ultimately mimicking native ECM.⁶

Stabilization of synthetic powders necessitates the use of binder chemicals which leave toxic and acidic residues in the printed construct.^{3, 54} Bio-ink, however, can be solidified using non-toxic methods.

Recently, Jang et al developed a vitamin B2 induced crosslinking and thermal gelation method to crosslink ECM and subsequently stabilize bio-ink layer-by-layer during the printing process.⁵⁵ Therefore, ECM powder use in 3DP potentially eliminates the need for toxic binders in stabilizing printed structures.

CLINICAL APPLICATIONS

ECM materials have numerous clinical applications in wound healing, surgical closure and reinforcement, and tissue reconstruction across large defects.⁵⁶ Unlike cell-based TE therapies, ECM-based therapies account for the difference in microenvironment between injured tissue and healthy tissue and thereby promotes sufficient vascularization and innervation for increased wound healing.⁵⁷ Therefore, ECM is efficacious in the treatment of wounds that are refractory to existing standard treatments and in patient populations, such as the elderly, who exhibit delayed wound healing.⁵⁸ Wounds that exhibit delayed healing and the inability to have complete closure benefit from constructive tissue remodeling promoted by ECM treatment. Moreover, the mechanical properties of ECM help to maintain wound closure and support surgical anastomosis.

ECM sheets or “patches” have been used to treat partial trachea defects,⁵⁹ volumetric muscle loss,⁶⁰ septal ulceration,⁶¹ diabetic foot ulcers,⁶² and cartilage defects;⁶³ to reinforce esophagojejunal anastomosis⁶⁴and esophageal hiatal hernia repair;⁶⁵ and to cover large defects from extensive surgical resection of cardiac tumors.^66-68 ECM powder, however, has a potentially broader clinical application because it can assume any shape and its solubilized form can be delivered by minimally invasive injection.

Dry powder

Commercially available ECM powder, most prevalent of which is pUBM, has been used as both a primary therapy in tissue regeneration and an adjunctive therapy.

Closure and healing of wounds that are traditionally difficult to treat such as diabetic ulcers and radiation wounds has been achieved with the topical application of pUBM.^{69, 70} Suture-less wound closure has been achieved with ECM powder in which powder particles coalesce to form a thin adhesive film.⁷¹ Suture-less wound closure is significant because suture filaments can act as a nidus for wound infection. As an adjunctive therapy, ECM powder has been used to salvage head and neck flaps in which graft imbibition and inosculation failed.⁷²

ECM powders have been used to bolster ECM sheet monotherapy treatment. The addition of powder treatment has been successful in treating refractory conditions such as pilonidal disease,⁷³ diabetic foot ulcers,⁷⁴ and primary and recurrent anal fistulas.⁷⁵ In particular, pilonidal disease is challenging to treat: post-operative sacrococcygeal intergluteal wounds require daily open packing with wound dressings over the course of several months and repeated flap surgeries. ECM powder in combination of ECM sheets accelerated wound healing and closure, eliminating the need for flap surgery and daily dressing changes.⁷⁶ Also, notably, ECM powder packing has provided novel and effective treatment for temporomandibular joint degeneration, which has never been successfully treated.⁷⁷

Solubilized powder

ECM powder can be solubilized subsequently undergo gelation to form liquid and gels, respectively. ECM scaffold implantation typically requires open surgery, but the fluidity of liquid and rheological properties of gels allow for minimally invasive clinical application.⁶ Depending on the viscosity of the liquid or hydrogels, they can be injected in vivo to promote tissue repair and regeneration. ECM hydrogel injections have successfully regenerated tendons, cartilage, and skeletal muscles.,^{16, 78–80} exhibiting potential for the treatment of tendinopathies, muscle loss, cartilage degeneration, and promote meniscus repair.

ECM particles can be used to repair areas of damage and prevent further pathological change. ECM particle emulsions injection offers potential in restoring functional myocardium and preventing maladaptive remodeling after ischemic events. Although emulsions contain insolubilized ECM, they are included in this section because they possess liquid properties that allows for injection. In the case of ischemic heart failure, treatments are typically limited to invasive surgical procedures. Myocardial infarction results in a transient upregulation of metalloproteinases that degrade native ECM, ultimately promoting fibrosis and scar formation. Treatment with ECM counteracts these changes. Intramyocardial injection of particulate cardiac ECM promoted growth of new tissue, angiogenesis, LV wall thickening, and improved cardiac function.⁸¹ ECM biomaterials have been used in the form of patches and intravascular catheter delivered liquid—but these do not penetrate the myocardium directly.⁸² ECM emulsions, however, can be injected through the epicardium and into the myocardium at the site of ischemia.

Temperature and ECM concentration can be modulated to induce changes in ECM form at a certain time. Control over these variables allows for novel clinical application of solubilized ECM powder. In a stroke model, solubilized ECM can be injected into brain where it is induced to form a hydrogel at body temperature. The initial liquid form allows for easy injection, permeation throughout peri-infarcted areas of the brain, and conformation to the contours of the brain. Transformation into the hydrogel form at body temperature, however, ensures that the ECM remains inside the stroke cavity once in the body. Injection of UBM in this manner has been successful in remodeling infarcted brain and supporting endogenous repair mechanisms of surrounding penumbra.⁸³ The same method has also been used in tendon regeneration in which the tendon ECM in liquid form was injected percutaneously and polymerized to form a fibrous hydrogel that was conducive for tendon regeneration.⁷⁸

CONCLUSION

ECM powder has novel applications in TE and RM, overcoming limitations of ECM scaffolds that have predetermined structure. Powder can be packed, spread, sprayed, or solubilized for minimally invasive delivery by injection. In both dry and solubilized forms, it conforms to the contours of tissue and fills areas of volumetric injury. Moreover, ECM powder and powder-derived constructs maintain their assumed shape, and most significantly, their size. Uncomminuted ECM, however, has the proclivity to contract over time and may eventually fail to cover or fill voids.

In tissue engineering, powder provides an ideal platform for ECM modification and 3D cell-matrix interactions. Powder is easily penetrated allowing for optimal diffusion of nutrients, maximal cell adhesion, and homogenous incorporation of reagents. In this way, effective 3D cell culture and bioconjugate reaction with growth factors, drugs, and molecular markers is achieved. As powder is conveniently modified, it also acts to modify. ECM powder is used to modify synthetic materials that have poor biocompatibility. This is clinically significant because ECM coatings mitigate inflammation and foreign body reactions to synthetic implantable surgical devices.

ECM powder also augments biocompatibility in 3D bioprinting: powder derived printing substrates can be stabilized by novel non-toxic means. Use of ECM powder derived substrates may potentially eliminate the need for toxic chemical binders used in standard solidification and stabilization protocols.

Clinically, ECM powder avoids the need for invasive surgical procedures, which are standard protocol for implantation of ECM scaffolds with fixed structure. Powder can be solubilized for injectable treatment. Injection is not only minimally invasive, but also capable of targeting areas of tissue injury that are difficult to access surgically.

In general, ECM is able to treat tissue injuries that heal slowly or are refractory to first-line therapies. ECM powder, however, has specifically shown promise as a therapy for previously untreatable tissue degeneration. The exact mechanism by which the powder form of ECM is a successful treatment where uncomminuted forms are not is unknown. Adjunctive therapy with ECM powder has rendered other ECM treatments successful in multiple models, suggesting that powder provides specific benefits that uncomminuted ECM lack.

The utility of ECM powder is a double-edged sword: advantageous properties that allow for novel application present challenges to further application. ECM powder and its solubilized derivatives can assume any shape; however, the destruction of the original 3D architecture that affords this may compromise the biological integrity of the ECM. Use of ECM powder in creating scaffolds provides incredible control over the construct’s characteristics. But, inherent in the ability to modulate characteristics and composition of powder-derived constructs is that any resulting construct will not ideally biomimetic.

Fabrication of ECM powder is challenging and highly susceptible to error at multiple levels. It is difficult to control particle size: powder is prone to agglomerating and cannot be effectively separated after initial comminution. Solubilization requires acidic conditions, which can elicit inflammation in vivo if not appropriately neutralized. Methods of sterilization are limited: aqueous solutions dissolve the powder. Problems at these key steps in fabrication need to be resolved for ECM powder to be used more extensively.

ECM powder may have greater potential as a biological therapy if more is known about the effect of comminution on ECM and if optimal fabrication protocols are determined. Further study on the modulation of ECM particles properties, may also inform for the design of tissue specific therapies.

Funding Statement

This work was supported by the No funding;

Abbreviations

3DP

3-Dimensional printing

e-beam

electron-beam

ECM

extracellular matrix

EDC

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride

GF

growth factor

iPSC

induced pluripotent stem cell

NHS

N-hydroxysuccinimide

PAA

peracetic acid

RM

regenerative medicine

SIS

small intestine submucosa

TE

tissue engineering

UBM

urinary bladder matrix

Article highlights

• Compared to their uncomminuted counterparts, ECM powders overcome challenges in and act as efficacious substrates for homogenous bioconjugate modification, 3D bioprinting, and treatment of intransigent tissue injury.

• Pulverization of ECM to a powder form affords structural flexibility that allows for novel applications in TE and RM.

• Of note, conformational adaptation to tissue contours and resistance to contraction enable ECM powder and solubilized derivatives effectively fill areas of tissue void.

• ECM powder can be uniformly incorporated with cells to optimize 3D cell culture and homogenously modified to effectively carry small molecules such as drugs and growth factors.

• Furthermore, ECM powder is used as a primary or adjunctive minimally invasive therapy by which it accelerates wound healing, facilitates complete wound closure, and salvages failed grafts.

Improving methods of powder fabrication, solubilization, sterilization, and crosslinking represents the most significant challenges to the effective and extended clinical use of ECM powders.