INTRODUCTION
In recent years, new and intriguing strategies aimed at enhancing the regenerative capabilities of stem cells during transplantation have been emerging. Among these, of particular interest are biomaterials capable of providing natural scaffolds to the transplanted, as well as (tissue) resident cells. In this editorial, we comment on the article that appeared in a recent issue of World J Stem Cells titled “High quality repair of osteochondral defects in rats using the extracellular matrix of antler stem cells”[1], in relation to other recent papers on similar topics. Furthermore, we aim to draw inspiration from this interesting subject for broader reflections, regarding stem cell niches and morphogenesis.
Adult stem cells, of mesenchymal origin in particular, have proven to be a promising possibility in the field of regenerative medicine, given their ability to differentiate into different cell types, as well as their ability to secrete trophic factors capable of modulating the trophism of recipient tissues, additionally showcasing a relevant immunomodulatory potential[2]. Many clinical trials are currently underway worldwide, and at the time of writing there are about 1500 clinical trials underway (https://clinicaltrials.gov/).
Despite the great potential, the beneficial effect derived from stem cell transplantation, mesenchymal stem cells (MSCs) in particular, appears to be moderate. Indeed, unmodified MSCs have shown limited advantages in various preclinical and clinical investigations, primarily attributable to challenges related to differences between MSCs derived from variegated sources, variability in culturing protocols, and, after transplantation, different engraftment efficiencies, inconsistent cell homing as well as cell viability[3]. All these issues prompted extensive research efforts over the past few decades to enhance cell functionality and potency.
In the past years, there has been exploration into different approaches to enhance the regenerative capabilities of stem cells. Various strategies have been tried, including cytokines and growth factors, hypoxia, pharmacological drugs, biomaterials, various culture conditions, a variety of biologically active molecules, and many others[4]. In this context, the utilization of scaffolds is particularly intriguing. The topic is of particular interest, as evidenced by the results of the PubMed search for the words “stem cell AND extracellular matrix”, that leads to 18010 results. However, if we circumvent the search by entering the words “animal-derived AND extracellular matrix” the results go down to 52, and they drop even further if keywords are “antler stem cell AND extracellular matrix”, that lead to 8 results, showing that this is still an under-explored field. Due to the limited number of publications, it is not only easier to grasp the state of the art, but we also consider this a clear indication of the necessity to delve into these emerging research domains.
BIOLOGICAL SCAFFOLD: A STRATEGY TO IMPROVE STEM CELL POTENTIAL
Considering the underwhelming outcomes in stem cell differentiation, it is reasonable to surmise that something pertaining specifically to the differentiation process might have been overlooked. The interaction between stem cells and their surrounding microenvironment plays a fundamental role in numerous processes, including cell migration, proliferation, lineage specificity, and tissue morphogenesis, thanks to the establishment of a conducive niche for the optimal expression of stem cell capabilities[5,6].
In vivo, the fate determination of stem cells is intricately regulated by a complex array of signals orchestrated within the cellular microenvironment. Within the stem cell niche, cell-to-cell signaling interactions are regulated by different components: Soluble factors from surrounding cells and/or tissues, the extracellular matrix (ECM) or cell substrate, the biophysical milieu, etc[7]. The sophisticated control mechanisms evolved by organisms to manage cell populations, including stem cells, are still largely unknown. Despite current gaps of knowledge, the importance of these structures remains unquestionably crucial.
Scaffolds not only can serve as platforms for in vitro cell growth before transplantation, but even more fascinating may be their direct transplantation in vivo, where they can serve as support for endogenous stem cells, which would go on to repopulate this type of structures. Indeed, a supportive microenvironment might be essential for regulating stem cell function, thereby activating or enhancing intrinsic host repair mechanisms[8]. There are several methods to investigate the chemical composition of the matrix: Biochemical analysis, scanning electron microscopy, transmission electron microscopy, infrared spectroscopy, raman spectroscopy, mass spectrometry, etc. Conversely, it remains much more difficult to study the mechanical properties, as well as the spatial interaction of structure proteins with colonizing cells. Despite this, it is clear that the mechano-sensitive pathways translate physical cues into biochemical signals, directing the cell towards a particular lineage[9].
Numerous efforts have been undertaken to comprehensively identify and characterize the influence of specific environmental cues on stem cell behavior, in order to achieve the most optimized support for cell growth and differentiation. Additionally, various materials, including both synthetic and natural ones, have been developed and, in many instances, already subjected to testing[10]. The inherent physicochemical characteristics of the biomaterial play a crucial role in cell behavior. Relevant properties include matrix stiffness[11-13], matrix porosity[14-16], topography, such as roughness and patterns[17-19], viscoelasticity[20,21], hydrophobicity[22], and surface charge[23]. Biomaterials used as support for cell growth are of different types, whether natural or synthetic, manufactured with different kinds of materials, such as ceramics, wood, and plastic, used alone or combined with living cells and tissues, also derived from animals[24].
Natural scaffolds are typically obtained through decellularization procedures, which are performed to eliminate cells and their components, particularly DNA and RNA, from the ECM. This process yields a natural matrix with preserved mechanical integrity[25]. Decellularized ECM (dECM) has been shown to be a viable type of natural scaffold for tissue engineering, as the ECM plays a critical role in tissue development. Studies for the possible use of decellularized scaffolds have been conducted since the 1950s[26]. In particular, the advent of dECM scaffolds offers a promising avenue in regenerative medicine, emulating an optimal non-immune environment, featuring native three-dimensional structures and a diverse array of bioactive components[27]. Decellularized scaffolds can be classified in several ways, one of which involves categorizing them into scaffolds derived from tissues and organs via decellularization, and scaffolds derived from matrix deposition by cells[28]. dECM scaffolds have been studied and applied in regenerative medicine for the repair or replacement of various tissues, e.g., skin, bone, heart, nerves, liver, lung, and kidney[27].
Drawing upon studies focused on the heart, an organ of particular interest in regenerative medicine due to the profound health implications of heart disease and the limited regenerative potential of its constituent cells, numerous investigations have been conducted since the 1990s. As early as 1999, a study documented the transplantation of decellularized pig-derived valves into sheep, revealing promising in vivo recellularization alongside the absence of calcification, a prognostically negative factor[29]. In the years that ensued, a multitude of studies have been published on this topic, collectively showcasing its feasibility, albeit without widespread adoption in clinical practice. This is due to issues regarding in vitro decellularization methods, as well as in vivo or ex vivo recellularization strategies[30].
An example of an interesting biomaterial derived from animal source is mentioned in the article we are taking into account. A field of possible scaffold massive clinical application is orthopedics, where prosthetics is widely used. The utilization of scaffolds capable of replacing or restoring damaged tissues holds great significance. It is precisely within this domain that biomaterials of various origins are under thorough investigation[31].
TISSUE SPECIFICITY OF ECM
This short roundup shows how stem cell fate is shaped by numerous factors and tangled interactions, through orchestrated engagement with soluble factors, neighboring cells, and ECMs: A localized biochemical and mechanical environment is established, characterized by intricate and dynamic regulatory patterning that stem cells perceive[32]. It is now clear that the capacity of stem cells to initiate differentiation into mature tissue cells is dependent on exposure to intrinsic properties of the ECM, determined by the chemical and protein composition, but also by the nature of the mechanical forces that the matrix is capable of generating[9], influenced, at least partially, by the origin of ECM itself.
Compelling experimental evidence has shown that ECM produced, even in vitro, by cells of different origins has very different properties. For example, ECM produced by stromal cells derived from human bone marrow (BM) and human adipose tissue (AD), BM-ECM and AD-ECM respectively, shows better abilities to induce proliferation in stem cells of equal origin, that is BM cells for the former and adipose derived cells for the latter. Additionally, to indicate further origin-dependent specificity, BM- and AD-ECM were found to selectively guide human MSC differentiation towards either osteogenic or adipogenic lineages, respectively, indicating tissue-specific effects of the ECM[33]. Even more interesting, is that ECM influenced cell morphology regardless of the human MSC origin, further supporting the notion of tissue specificity in the observed effects.
It is thus evident how the structures that specific cell types form are provided with a kind of memory (retained by the cells themselves), which also influences the cells that will later colonize those structures. In the same way, decellularized organs were shown to be substrates capable of promoting differentiation in the cell types they naturally harbored. The bio-instructive signaling cues found in dECM-based grafts can offer tissue-specific guidance for directing cellular behavior and coordinating cellular chemotaxis, as demonstrated in various applications: Regeneration of skeletal muscle[34], liver[35], trachea[36], and many others. There are already commercially available decellularized tissue-based products approved for clinical use, and, for instance, among these, heart valves and xenogeneic grafts made from bovine carotid arteries are the most commonly used[37].
Scientific evidence thus seems to suggest that ECM derived from cells with specific characteristics, including cancer, exhibits distinct properties. Much literature has paid attention regarding changes in ECM during cancer progression, demonstrating that dysregulation of ECM composition, structure, stiffness, and abundance contribute to invasiveness[38]. More intriguingly, ECM derived from healthy cells has the potential to yield beneficial effects in diseased contexts. ECM derived from human MSCs did not promote the proliferation of the cancer cell line HeLa, MCF-7, and MDA-MB-231[33]. When breast tumor cell lines, exhibiting various levels of invasiveness (benign, non-invasive, and invasive), were cultured in their own ECM or in ECM deposited by the other cell lines, they showed distinct cellular responses, correlating with the malignancy of the cell sources utilized in the ECM preparation. Accordingly, ECM derived from normal mammary gland cells were found to inhibit breast cancer proliferation[39]. As well, dECM obtained from normal lung tissue prompted apoptosis in MCF-7 cells, also inhibiting epithelial-mesenchymal transition, a common feature of malignancy[40].
THE IMPORTANCE OF ORIGIN
With the aim of identifying and studying mechanisms capable of inducing rapid tissue regeneration, the peculiar nature of deer antlers is of particular interest, as addressed in the commented article. Deer antlers are organs distinguishing them from other mammals, showing an incredible speed of growth, where rapid cell proliferation is elegantly controlled without resulting into malignancy[41]. Because of this feature, antler stem cells (ASCs) can serve as a model for examining the proteins and pathways implicated in maintaining a stem cell niche, as well as their activation and differentiation during organogenesis.
Animal derivatives are largely used in traditional medicine all over the world. The World Health Organization estimates that up to 80% of the global population (numbering over six billion people) primarily depend on medicines derived from animals and plants. For instance, in traditional Chinese medicine, over 1500 animal species have been documented for their medicinal applications[42]. The use of animal-derived products raises a number of issues, from hygiene to ethical ones. Certainly, the utilization of products that do not entail the sacrifice of animals is more ethically acceptable and thus preferable. Deer antlers, which are naturally shed by the animal each year, align with this expectation.
Deer antlers stand as the sole mammalian organ known to fully regenerate naturally once lost. In male deer, the presence of elevated testosterone levels in the bloodstream triggers the onset of antler formation during the second year of life. Antlers emerge as extensions from robust bony projections known as pedicles. Then follows a shift from pedicle to initial antler formation, and growth becomes evident as the integument covering the outgrowth transforms from regular skin (pedicle) to a distinct type of pelage, known as velvet. Due to a rise in circulating testosterone levels, antler growth stops, leading to a complete mineralization of antler bone, and to the shedding of the velvet covering. Following the season of pairings, when testosterone levels fall below this threshold, antlers are dropped (a process known as “antler casting”). In the following years, cycles of periodic regeneration and shedding of a new set of antlers from the pedicles begin[43].
Pedicles and initial antlers originate from a specialized periosteum, known as the antlerogenic periosteum (AP), which, if removed, prevents the formation of the stages. Later, annual antler regeneration is entirely reliant on the presence of cells in pedicle periosteum (PP) tissue. The rapid growth of an antler primarily occurs due to the activity of cells within the proliferation zone, specifically the reserve mesenchyme (RM), and cells within the RM must exhibit significant proliferation potential to sustain such rapid growth[44]. ASCs can be categorized into three types based on their source: Antlerogenic periosteal cells (APCs) originating from the AP, pedicle periosteal cells derived from the PP, and reserve mesenchymal cells (RMCs) originating from the RM[45].
The properties of the cells found in deer antlers, as well as the extracellular structures produced by them, display unique biological characteristics. In traditional Chinese medicine, deer antler extracts are considered to be of central importance in enhancing kidney function, fortifying tendons and bones, and extending longevity, among other purposes. Some scientific works have studied the inhibitory effect of Pilose antler extracts on cancer cells, showing interesting and promising results[46,47].
The cellular products of these cells also possess unique characteristics of clinical interest. Exosomes derived from ASCs are demonstrated to mitigate senescence in human MSCs in vitro[48], and significantly expedite the wound healing process, enhancing its quality, promoting the regeneration of cutaneous appendages (i.e., hair follicles and sebaceous glands), as well as improving the distribution pattern of collagen in the healed skin in a rat model[49]. In another study, it was demonstrated that ASC-derived exosomes were effective in alleviating the symptoms of pulmonary fibrosis in a mouse model system, while also increasing the survival rate of affected mice, in part through modulation of the immune response[50].
Several studies indicate that ASCs exhibit molecular traits akin to pluripotent and multipotent stem cells. This is evidenced, for example, by the fact that c-KIT (stem cell factor receptor) and Sca-1 (stem cell antigen-1), recognized markers of embryonic stem cells and tissue-specific stem cells, respectively, have been detected in over 70% of ASCs[51]. ASCs could be defined as MSCs with embryonic features. Indeed, ASCs express all typical MSC markers, including CD73, CD90, CD105, and STRO-1, along with certain markers associated with embryonic stem cells, such as Tert, Nestin, S100A4, nucleostemin, C-Myc, and Oct-4[52]. Negative expression markers were also assessed by cytofluorometry, in particular CD31, CD45, CD62p, CD133, and HLA-DR, that were absent or poorly expressed[50]. ASCs possess the capability, under particular culture conditions, to differentiate in osteogenic, chondrogenic and adipogenic lineages, as well as into muscle precursor cells and neuron-like cells, if isolated during a specific degree of differentiation[54]. These and other characteristics make ASCs a cell type with high potential for clinical applications.
In the paper commented on here, Wang et al[1] utilized a decellularized xenogeneic ECM derived from ASCs (RMC-ECM) as a substrate for MSC growth, providing evidence that supports the validity of this approach. Fascinatingly, RMC-ECM shows different regenerative properties when produced from cells in quiescent (APCs) or active (RMCs) state. Moreover, when transplanted in rats in order to repair osteochondral defects, ECM derived from deer stem cells, therefore of xenogeneic origin, performed better than allogeneic ECM produced by rat MSCs. The underlying reasons for this difference are still unclear, but some hypotheses can be discussed.
In vitro studies on antlers have effectively isolated and cultured unique ASCs discovered in the AP, PP, and RM, which play a pivotal role in tissue regeneration. Additionally, histological and morphological analyses have revealed the existence of various tissue types within the growth region of the antler[55]. The mechanism of antler regeneration has been extensively investigated, and it is believed that beyond a rise in systemic testosterone levels, a series of other signals from different hormones also intervene, i.e., estrogen, vitamin D, thyroid hormones, and cortisol[56], triggering the histogenesis of the pedicle and serving as the activation signal for the APCs located in the AP. The results shown in the annotated study demonstrate how RMC-ECM could replicate the proliferative effects, naturally shown in deer, in the restoration of osteochondral defects in other animal species by utilizing implantation of cell-free RMC-ECM sheets.
CONCLUSION
Many papers produced in recent years have sought to identify sources of efficient, and possibly cell-free, scaffolds that show high efficiencies and low costs, and that are worthy of exploration for potential applications in human health care. Evaluations of less traditional animal sources, such as deer, compared to those already widely studied such as pigs, may be of great interest for future clinical use.
Another consideration regarding this article, which from our perspective is even more fascinating, is the apparent confirmation that the data reported further supports the idea that the traits of an individual cell, the substances it generates, and the tissue it constitutes can evoke similar phenomena. This suggests a notion of ‘collective memory’ that permeates the entirety of an organism. Our perspective on this topic is that cells, along with all their cellular derivatives, inherently carry information about their own health status and have the capacity to transmit this information. Our group also studied the effects of an animal preparation on cancer human cells, in particular we demonstrated that extracellular vesicles derived from farm animal food derivatives (specifically pigs) can modulate human hepatic cell metabolism, thereby enhancing cell survival even in damaged contexts[57].
It almost seems as if there is a biological will to communicate one’s well-being (as well as any discomfort) to all that surrounds living organisms. This process is undoubtedly mediated by chemical factors, but they alone are not adequate to explain all aspects pertaining to this type of information transmission. There’s a memory that transcends chemistry’s boundaries, a phenomenon we can only define through mechanobiology. A relatively recent concept in the field is that of mechanical memory, which pertains to the enduring effects of mechanical stimuli long after their removal[58]. This memory that crosses the species boundary makes studies similar to the one commented on in the present editorial interesting, paving the way to a new vision of cellular biology where it appears to us that a greater harmony governed by more overarching natural laws may prevail.
Provenance and peer review: Invited article; Externally peer reviewed.
Peer-review model: Single blind
Corresponding Author’s Membership in Professional Societies: American Society for Biochemistry and Molecular Biology, 19046.
Specialty type: Cell and tissue engineering
Country of origin: Italy
Peer-review report’s classification
Scientific Quality: Grade A
Novelty: Grade A
Creativity or Innovation: Grade A
Scientific Significance: Grade A
P-Reviewer: Zhang W, China S-Editor: Wang JJ L-Editor: A P-Editor: Che XX
