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Decellularization

Decellularization is a biomaterial processing technique that involves the removal of cellular components, including nuclei and genetic material, from tissues or organs while preserving the structural, biochemical, and biomechanical properties of the extracellular matrix (ECM).^[1] This process yields acellular scaffolds that serve as biocompatible platforms in tissue engineering and regenerative medicine, mimicking the native tissue microenvironment to support cell adhesion, migration, proliferation, and differentiation with minimal immunogenicity.^[2] By addressing the global shortage of donor organs and tissues, decellularization enables the creation of personalized grafts that promote endogenous regeneration and reduce rejection risks compared to synthetic alternatives.^[3] The technique employs a combination of physical, chemical, and enzymatic methods tailored to the tissue type and desired scaffold properties.^[1] Physical approaches, such as freeze-thaw cycles, sonic disruption, or agitation, disrupt cell membranes mechanically, while chemical agents like detergents (e.g., sodium dodecyl sulfate or Triton X-100), acids, bases, or solvents solubilize cellular debris; enzymatic treatments using trypsin or nucleases further degrade proteins and DNA.^[2] For whole organs, perfusion-based decellularization—circulating agents through vascular networks—has become standard to maintain three-dimensional architecture, as demonstrated in protocols for hearts, livers, and lungs.^[3] Successful decellularization is rigorously evaluated using criteria from the American Society for Testing and Materials, including DNA content below 50 ng per mg dry ECM weight, absence of visible nuclei under staining, and retention of key ECM constituents like collagens, elastin, laminins, fibronectin, and glycosaminoglycans, alongside preserved mechanical strength.^[2] Originating from early studies on ECM isolation in the 1970s, decellularization gained prominence in the 1990s for applications in nerve grafts and bioprosthetic heart valves, with a pivotal 2006 review standardizing protocols for scaffold production.^[3] Key milestones include the 2008 perfusion decellularization of rat hearts, enabling repopulation with cardiomyocytes to restore partial function,^[4] and the first human implantation of a decellularized tracheal scaffold that same year.^[5] Today, decellularized ECM (dECM) scaffolds are applied in diverse clinical and research contexts, from wound healing dressings and vascular grafts to 3D bioprinting bioinks for cartilage, skin, and cardiac tissues, as well as disease models for cancer and drug screening.^[1] Challenges persist, including variability in ECM retention across methods and potential residual immunogenicity, driving ongoing innovations in sterilization and recellularization strategies.^[2]

Fundamentals

Definition and Principles

Decellularization is the process by which cellular components, including nuclei, cytoplasm, and other immunogenic elements such as DNA and RNA, are removed from tissues or organs, resulting in acellular scaffolds primarily composed of the extracellular matrix (ECM).^[6] This technique aims to produce biocompatible materials that minimize the risk of immune rejection while preserving the native structure and function of the ECM.^[7] The fundamental principles of decellularization revolve around the preservation of the ECM's intricate architecture and bioactive components, which serve as a natural scaffold for tissue regeneration. The ECM, consisting of proteins like collagen and elastin, glycosaminoglycans, and bound growth factors, provides essential structural support, biochemical signals, and biomechanical cues that guide cell adhesion, migration, proliferation, and differentiation.^[7]^[8] Effective decellularization seeks to retain these elements intact, ensuring the scaffold mimics the native tissue microenvironment to promote constructive remodeling upon implantation.^[7] Biologically, decellularization reduces immunogenicity by eliminating allogeneic or xenogeneic cellular antigens and residual DNA fragments that could trigger adverse host immune responses, thereby enabling the scaffolds' use in regenerative medicine applications across diverse species.^[7] This approach leverages the ECM's inherent biocompatibility to support host cell repopulation and tissue integration without eliciting chronic inflammation.^[9] Key prerequisites for successful decellularization include consideration of the source tissue's characteristics, such as cellular density and vascularization, which dictate the efficiency of cell removal; for instance, avascular tissues like cartilage may require milder treatments compared to highly vascularized organs to avoid compromising ECM integrity.^[7] Achieving an optimal balance between thorough cellular extraction and preservation of ECM components remains critical to maintaining the scaffold's mechanical properties and bioactivity.^[7]

Historical Development

The concept of decellularization emerged in the 1970s and 1980s amid efforts to overcome limitations in xenotransplantation and tissue preservation, driven by the need for biocompatible scaffolds that minimized immune rejection while retaining extracellular matrix (ECM) integrity.^[10] Early groundwork included the development of acellular vascular grafts, such as those explored by Malone et al. in 1983 using canine carotid arteries treated to remove cellular components.^[11] A pivotal advancement came from Stephen F. Badylak in the late 1980s, who pioneered the decellularization of porcine small intestinal submucosa (SIS) to create an ECM-based scaffold that supported host tissue remodeling without eliciting strong immune responses. This SIS material became the foundation for the first commercial decellularized products, including those from Cook Biotech, which received FDA clearance for applications like hernia repair in the early 2000s.^[12] The 1990s marked a shift toward standardized protocols, with increased emphasis on detergent-based methods to efficiently remove cellular antigens while preserving ECM structure.^[13] Perfusion techniques began to emerge for thicker tissues and whole organs, enabling more uniform decellularization and addressing challenges in simple immersion methods; early attempts included preliminary heart decellularization studies that laid the groundwork for organ-scale applications.^[14] A key regulatory milestone was the 1994 FDA approval of AlloDerm, a decellularized human dermal matrix used for burn treatment and soft tissue repair, validating the clinical viability of acellular scaffolds.^[15] In the 2000s, decellularization gained momentum through refined criteria for efficacy, as outlined by Gilbert et al. in 2006, who established guidelines requiring <50 ng dsDNA/mg dry weight and absence of visible nuclei to ensure minimal immunogenicity and structural preservation. This framework influenced subsequent protocols and facilitated broader adoption. Milestone achievements included Ott et al.'s 2008 demonstration of perfusion-decellularized rat hearts, producing acellular scaffolds with intact vascular architecture suitable for recellularization. Building on this, Petersen et al. in 2010 achieved successful recellularization of decellularized rat lungs with epithelial and endothelial cells, resulting in functional gas exchange upon transplantation, marking a breakthrough in whole-organ engineering. The 2010s and 2020s saw expansion to complex organs and integration with emerging technologies, including clinical trials for decellularized cardiac patches in the mid-2010s to treat myocardial infarction. Recent refinements have incorporated decellularized ECM into 3D bioprinting, as reviewed by Abaci and Guvendiren in 2020, enabling customizable bioinks that mimic native tissue environments for precise scaffold fabrication.^[16] Post-2020 developments include advanced decellularization methods for small-diameter vascular grafts and enhanced acellular ECM scaffolds for skin and organ regeneration, as detailed in reviews from 2023-2025.^[17]^[18] These developments underscore decellularization's evolution from basic tissue preservation to a cornerstone of regenerative medicine.

Methods of Decellularization

Overview of the Process

Decellularization begins with the procurement of source tissue or organ, typically obtained from allogeneic or xenogeneic donors under controlled conditions to ensure quality and minimize contamination.^[3] The subsequent general steps involve cell lysis to disrupt cellular membranes, removal of cellular debris through rinsing or flushing, extensive washing to eliminate residual components, and optional sterilization to render the extracellular matrix (ECM) acellular and biocompatible.^[1] These steps aim to produce a scaffold that retains the native ECM architecture while eliminating immunogenic cellular elements.^[3] Workflow variations depend on tissue characteristics, with immersion techniques commonly used for thin or avascular tissues such as skin, where the sample is submerged in processing solutions under agitation to facilitate uniform agent exposure.^[1] In contrast, perfusion methods are employed for thick, vascularized organs like the liver or heart, circulating solutions through the vascular network to ensure penetration into dense interiors and preserve three-dimensional structure.^[3] Tissue-specific factors, including density, vascularity, and compositional elements like collagen or glycosaminoglycans, guide protocol selection to achieve effective decellularization without compromising ECM integrity.^[1] The overarching goal is greater than 95% removal of cells alongside less than 50 ng double-stranded DNA per mg dry weight of ECM.^[3] Equipment such as bioreactors enables controlled perfusion for whole organs, while agitation systems support immersion processes by promoting solution distribution and debris expulsion.^[1] Following decellularization, the resulting scaffold can integrate with recellularization by serving as a substrate for cell seeding, though detailed repopulation strategies vary by application.^[3]

Physical Methods

Physical methods for decellularization rely on mechanical forces to induce cell lysis and removal while preserving the structural integrity of the extracellular matrix (ECM). These approaches apply physical stress to disrupt cell membranes without introducing chemical or enzymatic agents, making them suitable for tissues where biochemical alterations must be minimized. By generating mechanical disruptions such as ice crystal formation, pressure-induced denaturation, or vibrational energy, these techniques facilitate the release and clearance of cellular components, often through subsequent washing or perfusion.^[19] Freeze-thaw cycles represent a straightforward physical method, involving repeated freezing of tissue at low temperatures, typically -80°C, to form intracellular ice crystals that rupture cell membranes, followed by thawing to release cellular contents. Optimal protocols employ 3-5 cycles to achieve effective cell lysis while minimizing ECM damage, as excessive cycles can lead to fragmentation of matrix components. This technique has been particularly effective for decellularizing dense connective tissues like tendons, where it reduces residual nuclei to approximately 1% and DNA content by up to 80%.^[20] High hydrostatic pressure (HHP) applies uniform pressure, ranging from 200 to 1000 MPa, to denature cellular proteins and disrupt membranes, enabling cell removal via simple washing without compromising ECM ultrastructure. This method excels in preserving biomechanical properties and is widely used for avascular tissues such as bone and cornea, where pressures around 250 MPa for 10-60 minutes have demonstrated near-complete cell elimination while retaining collagen and glycosaminoglycans. Seminal work has shown HHP-treated blood vessels maintain biocompatibility for transplantation, highlighting its clinical potential.^[21]^[22] Sonication utilizes ultrasonic waves, typically at frequencies of 20-40 kHz, to generate cavitation bubbles that create transient pores in cell membranes, promoting lysis and enhancing penetration for debris removal. Electroporation complements this by delivering short electrical pulses (e.g., 100-1000 V/cm) to form reversible pores in cell membranes, facilitating targeted disruption in thicker tissues. Both techniques are advantageous for their non-invasive nature but require careful calibration to avoid excessive ECM shearing, with sonication often applied indirectly to mitigate structural alterations.^[23]^[19] Mechanical agitation involves physical forces like orbital shaking at 100-200 rpm or grinding for small tissue fragments, which dislodge cells through shear stress and improve exposure during washing. This method is ideal for thin or minced tissues, such as skin or cartilage slices, where it accelerates decellularization without specialized equipment. However, aggressive agitation can result in uneven cell removal or ECM disruption if not controlled.^[1] Overall, physical methods offer advantages in handling thick or dense tissues by providing mechanical disruption that penetrates deeply, often integrating with perfusion workflows for uniform processing. Their primary limitations include potential ECM fragmentation from over-application and incomplete debris clearance, necessitating optimization based on tissue type to balance efficacy and matrix preservation.^[24]^[25]

Chemical Methods

Chemical methods for decellularization primarily rely on detergents and solvents to solubilize cellular membranes and remove cellular components while aiming to preserve the extracellular matrix (ECM) structure and composition. These approaches disrupt lipid-protein interactions, denature proteins, and facilitate the extraction of nucleic acids and cytoplasmic debris, often through sequential application of agents at controlled concentrations and durations. Unlike physical methods, which use mechanical forces, chemical strategies target broad solubilization but require careful optimization to minimize ECM damage, such as glycosaminoglycan (GAG) loss or collagen denaturation.^[26]^[27] Ionic detergents, such as sodium dodecyl sulfate (SDS), are widely used for their strong lytic capacity. SDS at concentrations of 0.1-1% effectively solubilizes cell and nuclear membranes by denaturing proteins and disrupting lipid bilayers, achieving thorough cellular removal in dense tissues. However, higher concentrations (e.g., 1%) can cause significant ECM disruption, including up to 80% GAG loss and alteration of collagen fibril architecture, potentially compromising mechanical integrity.^[27]^[28] Sodium deoxycholate serves as a milder ionic alternative at 2-4%, solubilizing membranes with less protein denaturation and better retention of ECM fiber networks compared to SDS, making it suitable for applications requiring preserved bioactivity.^[27]^[19] Non-ionic detergents like Triton X-100 offer gentler solubilization, typically at 0.5-1%, by primarily breaking lipid-lipid and lipid-protein bonds without strongly affecting protein-protein interactions. This results in superior ECM preservation, with minimal GAG depletion (less than 20% in many cases) and maintenance of structural integrity, particularly beneficial for delicate tissues. Triton X-100 is less aggressive than ionic agents in removing antigenic components but excels in retaining growth factors and biomechanical properties.^[27]^[19]^[28] Acids and bases, along with chelating agents, complement detergents by inducing osmotic lysis or disrupting cell adhesions. Hypotonic solutions, such as distilled water or low-ionic-strength buffers, cause cellular swelling and lysis through osmotic shock, often applied initially for 12-24 hours to facilitate subsequent detergent penetration with minimal ECM impact. Bases like ammonium hydroxide (0.5-1%) hydrolyze nucleic acids and solubilize cytoplasmic contents via alkaline conditions, though prolonged exposure risks protein denaturation and should be monitored for pH (typically 8-10). EDTA, at 5-50 mM, chelates divalent cations like calcium to weaken cell-ECM adhesions, promoting detachment without direct ECM degradation when used briefly (e.g., 1-4 hours).^[19]^[1]^[28] Protocols for chemical decellularization generally involve sequential washes over 24-72 hours per agent, with agitation and multiple rinses in phosphate-buffered saline to remove residuals and prevent denaturation; pH is closely monitored (e.g., 7.4-8.0) to safeguard ECM proteins. For instance, a common sequence starts with hypotonic lysis and EDTA, followed by SDS or Triton X-100 incubation, and ends with nuclease washes, though the latter is enzymatic. Tissue-specific adaptations enhance efficacy: SDS (0.5-1%) via perfusion is preferred for robust organs like hearts to ensure deep penetration and complete lysis in myocardial tissue, while Triton X-100 (1%) is favored for vascular and neural structures to maintain tubular architecture and axonal guidance cues.^[26]^[27]^[28]

Enzymatic Methods

Enzymatic methods for decellularization employ biologically specific agents to selectively degrade cellular components while aiming to preserve the extracellular matrix (ECM). These approaches leverage enzymes that target proteins, nucleic acids, and other cellular residues, offering greater precision compared to broad chemical lysis agents.^[3]^[29] Proteases such as trypsin and dispase are commonly used to cleave cell-ECM adhesions and facilitate cell removal. Trypsin, typically applied at concentrations of 0.05-0.25% for 1-24 hours at 4-37°C, hydrolyzes peptide bonds on the carboxyl side of arginine and lysine residues, effectively disrupting cellular attachments but risking ECM degradation if exposure is prolonged.^[29]^[2] Dispase, often used at similar mild conditions (e.g., 1-2 hours at 37°C), specifically cleaves fibronectin and type IV collagen, enabling epidermal separation in skin tissues with minimal damage to deeper ECM structures like the basement membrane.^[3]^[2] Nucleases, including DNase I and RNase, target free DNA and RNA to mitigate immunogenicity post-decellularization. DNase I, administered at 50 U/mL for 1-3 hours at room temperature or 37°C, fragments DNA strands, reducing residual nucleic acids by up to 95% in protocols combined with washes.^[29] RNase complements this by degrading RNA under analogous conditions, ensuring comprehensive removal of genetic material that could elicit immune responses.^[3]^[2] Other enzymes like collagenase and hyaluronidase address specific ECM-associated components when partial modification is acceptable. Collagenase, used at low concentrations (e.g., 0.1% for 12-24 hours at 37°C), digests collagen fibers in dense tissues, though this may compromise scaffold integrity.^[29] Hyaluronidase breaks down glycosaminoglycans (GAGs) such as hyaluronic acid, applied for 24 hours at 37°C to enhance porosity without broad ECM loss.^[29] Protocols for enzymatic decellularization are tightly controlled, often involving sequential incubation at 37°C followed by extensive washes to eliminate debris and residual enzymes. For instance, a typical cycle might include trypsin treatment succeeded by nuclease application and PBS rinses over several hours.^[3]^[29] The primary advantage of enzymatic methods lies in their high specificity, allowing targeted removal of cellular elements while better retaining GAG content compared to detergent-based chemical approaches.^[3] However, limitations include the risk of over-digestion, which can disrupt ECM ultrastructure—such as basement membrane integrity—or leave enzyme residues that impair recellularization and provoke inflammation.^[2]^[29]

Verification and Evaluation

Standards for Successful Decellularization

Successful decellularization requires the efficient removal of cellular components while preserving the extracellular matrix (ECM) to minimize immunogenicity and maintain bioactivity for regenerative applications. Established criteria, formalized in seminal reviews such as Crapo et al. (2011), emphasize quantitative thresholds for cell removal and qualitative assessments of ECM integrity. These standards ensure that decellularized scaffolds are suitable for clinical translation by reducing the risk of adverse host responses. The American Society for Testing and Materials (ASTM) F3354-19 provides a standard guide for characterizing decellularized ECM scaffolds, outlining criteria for DNA content, ECM components, and mechanical properties.^[30]^[31] Core standards for cell removal include achieving greater than 95% elimination of cellular material, with residual double-stranded DNA (dsDNA) content below 50 ng per mg of dry tissue weight. Additionally, no visible nuclei should be detectable under microscopic examination using stains such as DAPI, and the absence of cytoplasmic remnants must be confirmed histologically to prevent inflammatory triggers. These thresholds, derived from comprehensive evaluations of decellularization outcomes, indicate effective clearance of immunogenic cellular debris.^[31] Biochemical criteria focus on retaining key ECM components to support cell adhesion, migration, and differentiation. Key components such as collagens, glycosaminoglycans (GAGs), and growth factors like vascular endothelial growth factor (VEGF) should be preserved to maintain structural integrity, hydration, signaling properties, and regenerative potential. These metrics highlight the balance between thorough decellularization and ECM preservation.^[1] Structural criteria require the maintenance of native tissue architecture, including fiber alignment and vascular networks, as verified through histological analysis. Disruption of these features can compromise mechanical properties and nutrient diffusion in recellularized constructs, underscoring the need for methods that avoid excessive ECM degradation. Preservation of such organization is essential for mimicking in vivo environments.^[31] To mitigate immunogenicity, residual DNA fragments must be shorter than 200 base pairs (bp), as longer fragments can activate Toll-like receptors and elicit innate immune responses. This threshold ensures that any remaining nucleic acids do not provoke chronic inflammation, a critical consideration for xenogeneic or allogeneic scaffolds.^[31] Regulatory guidelines from agencies like the FDA and EMA stress lot-to-lot consistency in decellularized scaffolds for clinical use, including reproducible cell removal, ECM composition, and biomechanical properties across batches. This consistency is vital for ensuring safety and efficacy in human applications, with guidance documents outlining qualification requirements for tissue-derived materials to address variability in sourcing and processing.^[32]

Analytical Techniques

Analytical techniques are essential for verifying the efficacy of decellularization processes by confirming the removal of cellular components while preserving the extracellular matrix (ECM) structure and composition. These methods encompass qualitative and quantitative assessments to ensure the resulting scaffolds are biocompatible and suitable for regenerative applications. Common approaches include histological, biochemical, biomechanical, imaging, and molecular analyses, each targeting specific aspects of decellularization success. Histological staining provides a qualitative evaluation of cell removal and ECM integrity. Hematoxylin and eosin (H&E) staining is widely used to confirm the absence of cellular nuclei and debris in decellularized tissues.^[33] 4-diamidino-2-phenylindole (DAPI) staining specifically detects residual DNA by binding to nucleic acids, allowing visualization of any remaining genetic material under fluorescence microscopy.^[34] Masson's trichrome staining highlights ECM components, particularly collagen fibers, to assess preservation of the matrix architecture without cellular remnants.^[35] Biochemical assays offer quantitative insights into key ECM constituents and residuals. The PicoGreen assay measures double-stranded DNA content, providing a sensitive indicator of decellularization completeness, often targeting thresholds below 50 ng DNA per mg dry tissue weight as referenced in established standards.^[36] The hydroxyproline assay quantifies collagen, a major ECM protein, by detecting its unique amino acid derivative after acid hydrolysis.^[37] Enzyme-linked immunosorbent assay (ELISA) is employed to evaluate glycosaminoglycans (GAGs) and residual cytokines, ensuring retention of bioactive molecules while confirming removal of immunogenic factors.^[38] Biomechanical testing evaluates the functional integrity of the decellularized ECM by assessing mechanical properties akin to native tissues. Uniaxial tensile testing measures tensile strength and elasticity, revealing how decellularization affects stiffness and load-bearing capacity, with preserved scaffolds often exhibiting moduli comparable to native counterparts.^[39] Advanced imaging techniques visualize microstructural details at high resolution. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) examine ultrastructure, confirming the retention of nanofibrous networks and pore architecture post-decellularization.^[40] Confocal microscopy enables three-dimensional (3D) reconstruction of ECM architecture, highlighting spatial organization and any disruptions in fiber alignment.^[41] Molecular analysis delves into compositional profiles at the proteomic and genomic levels. Mass spectrometry-based proteomics identifies ECM protein composition, detecting retained collagens, laminins, and proteoglycans while quantifying changes due to processing.^[42] Quantitative polymerase chain reaction (qPCR) detects residual nucleic acids, offering high sensitivity for fragmented DNA or RNA that may evade other assays.^[43] Recent advancements include non-destructive and automated methods for improved evaluation. Raman spectroscopy provides label-free, non-invasive assessment of ECM preservation by analyzing molecular vibrations, enabling in situ quantification of proteins and lipids without tissue disruption.^[44] AI-based image analysis enhances consistency in histological assessments, using machine learning algorithms to automate quantification of cellular remnants and ECM features from microscopy images, reducing subjectivity in quality control.^[45]

Applications in Regenerative Medicine

Tissue Engineering Scaffolds

Decellularized extracellular matrix (dECM) scaffolds have emerged as a cornerstone in tissue engineering, providing biocompatible frameworks that mimic the native tissue environment to support repair and regeneration. These scaffolds are fabricated from various decellularized tissues, preserving the structural and biochemical components of the extracellular matrix while removing cellular elements to minimize immunogenicity. For instance, decellularized human dermis, such as AlloDerm, is widely used as a dermal substitute for wound healing, promoting re-epithelialization and tissue integration in full-thickness skin defects.^[46] Similarly, cartilage-derived dECM matrices serve as scaffolds for joint repair, offering mechanical support and chondrogenic cues to facilitate cartilage regeneration in osteoarthritic models. Customization of dECM scaffolds enhances their adaptability for specific applications, particularly through processing techniques that enable advanced fabrication methods. Cryomilling decellularizes and pulverizes ECM into fine powders or hydrogels, which can be formulated as bio-inks for 3D bioprinting at concentrations typically ranging from 1-5% dECM to balance printability, viscosity, and bioactivity. This approach allows for the creation of patient-specific constructs, such as layered cartilage scaffolds or vascularized skin equivalents, by combining dECM with supportive polymers like alginate or gelatin. The mechanisms underlying dECM scaffold efficacy involve the delivery of bioactive cues that guide host cell behavior. Retained components, including collagens, glycosaminoglycans, and growth factors like TGF-β, promote cell infiltration, adhesion, proliferation, and extracellular matrix remodeling, fostering constructive tissue formation rather than scar deposition.^[47] Compared to synthetic scaffolds, dECM offers superior biocompatibility and site-specific bioactivity, reducing foreign body responses and enhancing long-term integration due to its natural architecture and signaling molecules.^[48] Preclinical studies demonstrate the translational potential of these scaffolds. Porcine small intestinal submucosa (SIS) has been effectively used to augment rotator cuff repairs in rat models, where it acts as an inductive scaffold promoting tendon regeneration and improving biomechanical strength without adverse immune reactions. Likewise, ovine urinary bladder matrix scaffolds support esophageal reconstruction in large animal models, enabling epithelial and muscular remodeling to restore functional continuity.

Whole Organ Decellularization and Recellularization

Whole organ decellularization involves perfusing intact organs with detergents to remove cellular components while preserving the extracellular matrix (ECM) architecture, including vascular networks. For hearts, protocols typically employ antegrade or retrograde perfusion through the aorta or vena cava using 1-5% sodium dodecyl sulfate (SDS) at controlled pressures over 4-12 days depending on organ size, which effectively clears >99% of DNA and cells while retaining collagen, elastin, and glycosaminoglycans.^[49] Similar perfusion strategies apply to other organs; for lungs, cycles of SDS (0.1-1%) followed by CHAPS or Triton X-100 are perfused via pulmonary arteries and veins for 24-72 hours to maintain alveolar and vascular integrity. These methods, pioneered in rodent models and scaled to porcine tissues, ensure scaffold biocompatibility and mechanical properties akin to native organs.^[49] Recellularization aims to repopulate these acellular scaffolds with appropriate cell types to restore function, often using bioreactor systems that mimic physiological conditions. Initial seeding focuses on endothelial cells (e.g., human umbilical vein endothelial cells or induced endothelial cells) perfused through the vasculature to line vessels and prevent thrombosis, followed by infusion of parenchymal cells such as cardiomyocytes, hepatocytes, or induced pluripotent stem cell (iPSC)-derived progenitors into the interstitial spaces. Perfusion culture in bioreactors for 7-14 days promotes cell adhesion, proliferation, and ECM remodeling, with dynamic flow enhancing nutrient delivery and waste removal.^[50] This sequential approach has yielded partial functional recovery, such as synchronized contractions in recellularized rat hearts generating up to 2% of native pressure.^[4] Notable examples include decellularized rat livers recellularized with primary hepatocytes via portal vein and bile duct infusion, demonstrating albumin synthesis and functional bile production for up to 5 days in vitro.^[51] In porcine kidney models, scaffolds decellularized with SDS and recellularized with renal epithelial and endothelial cells have supported glomerular filtration rates comparable to partial dialysis function when transplanted into pigs, filtering blood and producing urine-like filtrate.^[52] These proofs-of-concept highlight the potential for organ-specific regeneration but underscore scaling challenges, including retention of vascular patency to avoid thrombosis and achieving uniform cell distribution across complex 3D architectures, which often results in heterogeneous repopulation and limited long-term viability. Recent advances in 2024 have integrated decellularized ECM (dECM) hydrogels derived from whole organs into organoid culture, enabling the formation of vascularized mini-organs like liver buds that exhibit improved self-organization and metabolic function compared to traditional Matrigel-based models.^[53] These dECM-supported organoids, seeded with iPSC-derived hepatic and endothelial cells, better recapitulate native zonal architecture and drug responses, bridging whole-organ engineering with scalable miniature systems.^[53]

Clinical and Preclinical Examples

Preclinical studies have demonstrated the potential of decellularized extracellular matrix (dECM) in organ transplantation models. In a landmark experiment, decellularized rat lungs were recellularized with primary rat lung endothelial and epithelial cells before heterotopic transplantation into recipient rats, where the engineered lungs supported gas exchange for up to 2 hours, indicating preserved vascular and alveolar functionality.^[54] Decellularized porcine-derived scaffolds have shown promise in cardiovascular repair models. For instance, in sheep models of myocardial infarction, patches made from decellularized porcine small intestinal submucosa were applied to the infarcted area, resulting in improved left ventricular ejection fraction compared to untreated controls, alongside enhanced neovascularization and reduced scar tissue formation. Recent preclinical work has explored dECM for reproductive tissue engineering. In sheep models, decellularized uterine scaffolds were used to create bioengineered patches that replaced full-thickness uterine defects; these grafts integrated well, promoted tissue regeneration, and restored structural integrity, with potential implications for fertility restoration in uterine injury models.^[55] Clinically, decellularized porcine heart valves, such as the Matrix P bioprosthesis introduced in the late 1990s, have been widely implanted for valve replacement, exhibiting lower rates of calcification compared to glutaraldehyde-fixed valves due to the removal of cellular antigens.^[56] These valves demonstrate long-term patency. Decellularized dermal matrices, like Integra, have been successfully used as grafts for severe burn wounds, achieving median take rates of 95% when combined with autologous skin grafts, facilitating faster wound closure and improved cosmetic outcomes over traditional treatments.^[57] Human trials of dECM-based cardiac patches for myocardial infarction have advanced into early phases in the 2020s. In a first-in-human study, a decellularized human pericardial ECM scaffold (PeriCord) seeded with stem cells was epicardially applied to patients with ischemic cardiomyopathy, demonstrating safety and feasibility in phase I evaluation with no major adverse events reported at short-term follow-up.^[58] Overall, decellularized materials in these applications have led to reduced immune rejection and inflammation compared to cellularized xenografts, with preclinical and clinical data showing decreased inflammatory cell infiltration and lower rates of graft failure.^[59]

Challenges and Future Perspectives

Limitations and Risks

Decellularization processes face significant technical limitations, primarily stemming from challenges in achieving complete cell removal while preserving the extracellular matrix (ECM). Incomplete decellularization often leaves residual cellular components, such as DNA fragments exceeding 50 ng/mg dry weight or longer than 200 bp, which can elicit immunogenicity and provoke adverse host responses.^[31] Harsh chemical agents, like ionic detergents (e.g., sodium dodecyl sulfate), effectively eliminate cells but damage ECM architecture, including disruption of collagen fibers and significant loss of glycosaminoglycans (GAGs), typically ranging from 40-70% in treated tissues. These alterations compromise the scaffold's biomechanical properties and bioactivity, potentially hindering tissue regeneration. Biological risks associated with decellularized scaffolds include the potential for pathogen transmission, particularly when donor screening is inadequate. Without rigorous specific-pathogen-free (SPF) protocols for source tissues, latent viruses or bacteria may persist in the ECM, posing transmission risks to recipients in clinical applications. Additionally, decellularized vascular scaffolds exhibit heightened thrombogenicity due to exposed subendothelial collagen and incomplete preservation of anticoagulant factors, leading to clot formation upon implantation if not properly endothelialized. Ethical concerns also arise in tissue sourcing, especially for xenogeneic materials, where animal welfare and donor consent protocols must align with emerging standards to mitigate controversies.^[60] Scalability remains a major hurdle, driven by high production costs due to specialized equipment and reagents—and inherent variability in donor tissues. Differences in age, health, and species of origin result in inconsistent ECM composition and mechanical strength, complicating reproducible outcomes across batches. Immune responses further complicate use, especially with xenogeneic sources where residual alpha-gal epitopes trigger low-level antibody-mediated reactions, even after processing. Chronic inflammation has also been observed in some implants, linked to damage-associated molecular patterns (DAMPs) released from altered ECM. Decellularization challenges include risks such as excessive degradation during enzymatic or chemical treatments, which can cause scaffold collapse by damaging structural proteins like collagen.^[61] Incomplete recellularization exacerbates this, often resulting in thrombosis within vascularized constructs due to poor endothelial coverage and persistent pro-coagulant surfaces.^[61] Adherence to established standards for DNA quantification and ECM integrity assessment can help identify these issues early, though they do not eliminate the underlying risks.^[31]

Emerging Trends and Regulatory Considerations

Recent advancements in decellularization have integrated CRISPR gene editing technologies with recellularization processes to enhance compatibility and functionality of decellularized extracellular matrix (dECM) scaffolds. By editing patient-derived cells, such as induced pluripotent stem cells, to remove immunogenic factors or introduce therapeutic genes, researchers aim to minimize rejection risks in personalized tissue constructs.^[62] This approach has shown promise in preclinical models, where CRISPR-modified cells seeded onto dECM promote vascularization and tissue maturation without eliciting strong immune responses.^[63] dECM materials are increasingly incorporated into organ-on-a-chip systems and organoid cultures to better recapitulate native tissue microenvironments. In 2024 reviews, dECM-derived hydrogels have been highlighted for supporting multicellular interactions in liver and lung organoids, improving drug testing accuracy by preserving bioactive cues like growth factors and stiffness gradients.^[64] Similarly, AI-driven optimization of decellularization protocols has emerged, using machine learning algorithms to predict and refine parameters such as detergent concentration and perfusion time for personalized scaffold production based on tissue source variability.^[65] These computational models reduce trial-and-error, achieving up to 95% efficiency in ECM retention while minimizing damage.^[66] Hybrid dECM-synthetic composites represent a key trend in enhancing mechanical properties for load-bearing applications. Combining dECM with polymers like gelatin methacryloyl (GelMA) or alginate creates scaffolds with tunable elasticity and bioactivity, outperforming pure dECM in durability for bone and cartilage repair.^[67] In 3D bioprinting, dECM-based bioinks have advanced neural tissue engineering; for instance, brain-derived dECM inks support neural stem cell differentiation into mature neurons, with axon outgrowth rates 2-3 times higher than synthetic alternatives, as demonstrated in peripheral nerve models.^[68] These bioinks exhibit shear-thinning behavior, enabling high-resolution printing of complex neural architectures.^[69] Regulatory frameworks are evolving to facilitate clinical translation of dECM products. The U.S. Food and Drug Administration's (FDA) Regenerative Medicine Advanced Therapy (RMAT) designation, outlined in 2025 draft guidance, accelerates approval for dECM scaffolds addressing serious conditions by allowing surrogate endpoints and priority review, provided preliminary evidence shows potential to meet unmet needs.^[70] This applies to tissue-engineered products where dECM serves as the primary bioactive component. The European Medicines Agency (EMA) emphasizes potency assays in guidelines for advanced therapy medicinal products (ATMPs), requiring validated tests to confirm dECM bioactivity, such as cell adhesion and growth factor release, to ensure consistent therapeutic performance.^[71] Looking ahead, human clinical trials for whole-organ decellularization, particularly liver, are projected to advance toward feasibility by the early 2030s, building on preclinical successes in porcine models that demonstrate functional engraftment and bile production.^[72] Standardization efforts, including proposed ISO guidelines for bioprocessing decellularized ECM of soft tissues, aim to establish uniform criteria for DNA removal and mechanical integrity by 2025, promoting reproducibility across labs.^[73] Recent safety reviews from 2023-2025 underscore the need for long-term immunogenicity testing, recommending assays for residual antigens like α-Gal and monitoring immune activation beyond 6 months post-implantation to mitigate chronic rejection risks in clinical settings.^[74]

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Recent advances in decellularized biomaterials for wound healing
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