Slaughterhouse waste: a unique and sustainable source for dECM-based bioinks

End-stage tissue and organ failure, stemming from an array of factors such as diseases, injuries, and developmental anomalies, has evolved into major economic and healthcare problems. Currently, standard clinical approaches to tackle these issues rely on organ donation. However, the shortage of donors and growing recipient pools limit the reliance on transplantation [1]. In fact, back in 2011, studies estimated that merely a third of patients in need of transplantation would receive one, and the overall likelihood of someone needing a transplant far outweighed their ability to become a donor [2]. Sadly, modern-day statistics fail to provide a better outlook. The rising incidences of debilitating conditions, along with substantial logistical constraints that affect successful transplantation, support trends pointing to a growing mismatch between tissue/organ supply and demand. These pervasive issues emphasize the need for alternative solutions to tackle the substantial global shortage.

To address this pressing medical necessity, tissue engineering approaches have emerged as viable solutions. These multidisciplinary fields combine knowledge and technologies from a diverse spectrum of areas, including biology, chemistry, engineering, medicine, pharmacology, materials science and, more recently, artificial intelligence [3]. Their primary goal is to develop treatments and innovations for repairing or replacing damaged tissues and organs [4]. Tissue engineering employs a wide range of techniques to design and create artificial/bio-artificial constructs. This process involves assembling biomimetic materials encompassing scaffolds, cells and signaling molecules to replicate natural tissue architectures and functions, thereby offering promising solutions to meet the growing demand for transplantation [5].

One of the most promising techniques within the field of tissue engineering is 3D bioprinting because of its versatility, ease of use, and precision of the fabrication process. 3D bioprinting, also known as additive manufacturing, is a process of joining materials layer-by-layer to build a framework that mimics native tissue architectures. This innovative approach has many applications, such as generating skin grafts for burn victims, cartilage and bone replacements for orthopedic procedures, nervous system repair, cornea replacement and vascularized tissues [6].

A key component of adaptive manufacturing is the bioink, which is a specialized natural or synthetic material infused with living cells, growth factors, and other bioactive compounds. A bioink should possess desired physicochemical properties, such as proper biomechanical, rheological, and biochemical characteristics of the target tissues. They are composed of a variety of materials, including natural polymers such as alginate and collagen, synthetic polymers like polyethylene glycol (PEG), and even the decellularized extracellular matrix (dECM) [7].

The dECM is typically derived from cadaveric sources, as well as laboratory bovine, ovine, murine, porcine, and simian tissues that share anatomical and physiological similarities with humans, including. In recent years, dECM components have been used in models for biomaterials-based biofabrication [8]. These dECM components can be homogenized into solutions containing bioactive cues that recapitulate a natural cellular environment. Such an environment will synergistically provide physical barriers, anchorage sites, and pathways for cellular growth, migration, and differentiation – essential for morphogenesis and a basic set of fundamental criteria for designing functional human tissues and organs.

We recognize the untapped potential of dECM derivatives found in animal tissues from livestock. Domestically raised animals, which are routinely slaughtered for food production, contribute to approximately 150 million tonnes of organic waste annually. This waste is notably rich in key bioink components like collagen, elastin, fibronectin, and hyaluronic acid [9]. As such, repurposing slaughterhouse waste offers a viable and sustainable method for producing hydrogels, tailored to specific tissue needs and cost-effective in nature. These hydrogels are adept at encapsulating desired cells and can be cross-linked or stabilized during or immediately after the bioprinting process, which is vital for achieving the precise shape, structure and architecture of the intended bioprinted construct [10].

This commentary underscores the significant aspects of developing bioinks from slaughterhouse waste. It highlights the abundance of available starter materials, outlines formulation and classification techniques, and discusses the derivation of these materials from discarded dECM sources. The repurposing of such waste not only presents a solution to an environmental problem but also unlocks new possibilities in the realm of tissue engineering and regenerative medicine.

A vast supply of starter materials for bioink production

Despite advancements in bioprinting technologies, finding suitable bioinks that align with the necessary mechanical, rheological, and biological requirements has remained a challenge to date [11]. The repurposing of slaughterhouse waste presents a promising solution with several advantages, offering a vast supply of tissues and organs. This organic waste from abattoirs is an abundant source that is ideal for biofabrication, supplementing laboratory and cadaveric sources.

Financially, utilizing discarded slaughterhouse waste for bioinks is cost-effective compared with current market sources, ranging from $400–1000 for just 3 to 4 ml [12]. This affordability enhances the potential of this method for research and medical applications, providing researchers with ample raw materials for experimentation.

However, this approach has several drawbacks as it requires rigorous sterilization to mitigate pathogen and toxin contamination, thorough quality control throughout the production process [13], and comprehensive biocompatibility testing [14]. Despite these challenges, repurposing abattoir tissues/organs for bioinks reduces waste and aligns with the circular economy model. It transforms slaughterhouse byproducts into valuable resources for the biomedical field, offering a sustainable and innovative solution to current bioprinting challenges.

With global meat consumption escalating due to rising income levels and population growth, the meat industry generates 150 million tonnes of organic waste annually [10]. This waste predominantly comprises 50–60% of the discarded weights of bovine, ovine, and porcine livestock. Some of these remains are repurposed to create high-value products like animal feeds, fats and oils, fertilizers and biogas [9]. However, a significant amount of this waste is left unutilized, primarily due to cultural, religious, and health considerations. The conventional disposal methods for this waste, including landfilling, incineration and burial, pose substantial environmental challenges [15].

To help address the significant waste in the meat industry, researchers are focused on transforming slaughterhouse byproducts into valuable biomaterials and scaffolds for use in regenerative medicine and adaptive manufacturing. This conversion of byproducts into bioinks, as opposed to traditional disposal methods, offers a range of unique advantages for bioprinting. These include sustainability through waste repurposing, efficient utilization of resources and cost–effectiveness. Additionally, these bioinks possess inherent biocompatibility ideal for tissue engineering, allow for customized formulations tailored to specific applications, and reduce reliance on animal-derived resources. Moreover, this approach stimulates innovation in materials science and aligns with the principles of waste reduction and resource optimization central to a circular economy.

All of these advantages collectively can contribute to developing a comprehensive catalog of bioinks, each meticulously optimized for constructing cellular microenvironments. These microenvironments are designed to deliver precise biochemical and biomechanical cues critical for cellular functions. Such cues include cell surface recognition, growth, chemotaxis, and responses to mechanical stimuli such as shear stress, stretching, and compression. Additionally, they can mimic the natural matrix's elasticity and rigidity, ensuring the bioinks are tailored to meet the requirements of tissue-specific applications.

Bioink formulation & classification techniques

The bioink, an essential element in adaptive manufacturing technologies, is typically a hydrogel-based solution comprising one or more biomaterials [16,17]. In the bioprinting process, this solution encapsulates living cells, growth factors, and other vital biomolecules, including extracellular matrix components, which are integral for creating functional and mechanically robust constructs [11]. The bioink must exhibit specific biomaterial properties to ensure the printed tissues and organs possess the desired characteristics. These properties include printability, mechanical strength, biodegradability, and modifiable functional groups [18].

Printability is influenced by factors such as the viscosity of the solution, surface tension of the bioink, and its ability for self-crosslinking, which is critical for maintaining the integrity of the 3D structure. The hydrophilic nature and viscosity of the bioink also play a significant role in the reliability of the printing process and the effective encapsulation of live cells. Controlled gelation and stabilization are crucial; these refer to the bioink's transition from a liquid to a gel-like state, a process that must be precise and maintained throughout the printing. Furthermore, bioinks must meet biological standards, including biocompatibility and cytocompatibility, ensuring they are safe for living cells and tissues and can support cell survival, proliferation and functionality during and post-printing.

Bioinks can be broadly classified into natural and synthetic categories based on composition. Natural bioinks (collagen, agarose, gelatin, alginic acid, hyaluronic acid, chitosan, etc.) have central roles as bioinks for 3D printing tissues and organs due to their ability to mimic the structure of the ECM and their biocompatibility and biodegradability. On the other hand, synthetic bioinks are engineered using artificial polymers like polyethylene glycol (PEG), poly(L-lactic) acid (PLA), poly(lactic-co-glycolic) acid (PLGA) and polycaprolactone (PCL) [19]. While they offer properties such as controllability of mechanical stability, degree of photo crosslinking, pH, and temperature responsivity, which are not present in natural bioinks, synthetic bioinks represent only approximately 10% of the bioinks used in 3D printing [20]. This is because they lack sites for cellular recognition and proliferation, along with the challenges regarding the encapsulation of cells.

These solutions can also be categorized as scaffolding or non-scaffolding based on whether they contain a supporting structure that provides cells within the printed construct. While scaffold-based bioinks provide a supportive framework that mimics the ECM, scaffold-free bioinks rely on cell–cell interactions and self-assembly to form a desired tissue. Scaffold-free bioprinting is viewed as a promising approach in tissue engineering because it allows for the replication of native tissues in a shorter timeframe compared with the more conventional method of using bioinks containing cells. These two distinctions play a big role in the applications of bioinks for 3D printing, tissue engineering, and regenerative medicine.

Bioink derivation from discarded dECM sources

From these wastes, it is possible to generate tissue-specific and ECM-based bioinks through a technique known as decellularization after unwanted components like fat are removed from the native tissue. Decellularization is the process of removing cellular and nuclear matter from the native tissue using several methods, such as chemical, physical and biological treatments, while minimizing any adverse effects on the composition, biological activity and mechanical integrity of the residual ECM. The first step in preparing bioinks is harvesting tissues from the slaughterhouse waste and then effectively eliminating cells and a minimum of 90% of the native DNA. The decellularization process varies based on tissue type, as well as factors like tissue density, lipid content, and ECM composition. Residual decellularization agents must be removed to prevent cytotoxicity, followed by sterilization with agents like peracetic acid or ethanol. The tissue is then lyophilized and pulverized into small particles using instruments like a cryomill or homogenizer.

The resulting dECM powder can be solubilized using pepsin in an acidic environment, with pH adjustment to stop digestion followed by gelation at 37°C [21]. The ECM is an intricate network composed of an array of multidomain macromolecules organized in a cell-specific manner. It also acts as a dynamic repository for growth factors and bioactive molecules, profoundly influencing fundamental cellular behaviors such as proliferation, adhesion, migration, polarity, differentiation and apoptosis. Major components include collagens, proteoglycans, elastin and cell-binding glycoproteins, each with distinct physical and biochemical properties [22].

The unique properties of dECM-based bioinks are seen as central to their role in revolutionizing tissue engineering and regenerative medicine. The blend of collagens, proteoglycans, glycoproteins, and bioactive molecules contained within the dECM bioinks ensures both biocompatibility and biomimicry. Since these tissues retain the microenvironment, they promote cell adhesion and foster tissue regeneration via structures such as collagen and fibronectin. Furthermore, their biodegradability stems from the composition of structural molecules, ensuring they are naturally absorbed over time [23]. Their remarkable versatility and ability to be precisely tailored for tissue-specific formulations set dECM-based bioinks apart from other inks used in adaptive manufacturing. Another key element is rheological characterization, which ensures the printability of these bioinks. While components such as glycosaminoglycans and proteoglycans ensure hydration of the ECM and endure compressive forces, fibrous matrix proteins such as collagen and laminin provide resistance to tensile forces [24]. This combination entails significant variations of rheological properties related to low viscosity or poor consistency that translates into limited printability. Consequently, this is associated with poor intrinsic mechanical properties such as low modulus, viscoelasticity and yield stress [25].

To solve these problems, researchers use a framework printed with high mechanical strength biomaterials like PCL or silicone rubber, which can be used to maintain the structure of the dECM. Another effective strategy is to combine the dECM with other synthetic polymers or active molecules [26]. For instance, a hydrogel bioink containing porcine cardiac acellular extracellular matrix (cdECM), Laponite-XLG nanoclay and poly (ethylene glycol)-diacrylate (PEG-DA) components exhibited more shear-thinning behavior making the bioink suitable for extrusion through bioprinter nozzles during the printing process. Importantly, it quickly regains its original viscosity after reducing shear stress, ensuring structural integrity and cell viability. This also enabled it to support cell-laden up to 7 days post-print, indicating that it could withstand shear conditions [25].

Nevertheless, there exist several obstacles in the widespread adoption of dECM-based bioinks. These challenges encompass issues like inconsistent and non-standardized decellularization procedures, limited control over the printability and mechanical stability of dECM bioinks [19], and concerns related to immune responses, which could pose substantial difficulties in ensuring long-term in vivo safety. In addition to these biological challenges, the impediments related to large-scale production, influenced by the tissue-specific nature of dECM from various sources, require a long time, making the products costly. Ethical considerations also play pivotal roles in shaping the progress and implementation of dECM-derived bioinks [7]. To this end, further research is required to optimize protocols, refine characterization strategies, and assess long-term functionality to advance bioinks derived from slaughterhouse waste.

Conclusion

Regenerative medicine strategies, like tissue engineering, have emerged as transformative fields in healthcare and biotechnology, potentially revolutionizing our approach to the shortage of organs available for transplantation. Innovative solutions are essential given the critical scarcity of organ donors and the continually expanding list of individuals awaiting life-saving transplants. Among various methods, 3D bioprinting is an advantageous technique for fabricating tissue engineering scaffolds, with bioinks being a pivotal component. Biomaterials discarded from slaughterhouses can be repurposed to generate tissue-specific bioinks derived from acellular tissue matrices. These bioinks are prized for their biocompatibility, biodegradability, and ability to support cell attachment and proliferation, marking them as promising candidates for tissue engineering applications. Still, further research is required to enhance production standards that support adequate structural and functional capacities needed for the long-term viability of slaughterhouse-sourced bioinks.