Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

Exploitation of herpesvirus immune evasion strategies to modify the immunogenicity of human mesenchymal stem cell transplants

Background

Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected.

Methodology/Principal Findings

We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients'' natural killer (NK) cells were depleted prior to cell transplantation.

Conclusions/Significance

Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable.     

Transdifferentiation of mesenchymal stem cells derived from human fetal lung to hepatocyte-like cells

The great shortage of human hepatic cells makes it desirable to generate extrahepatic stem or precursor cells. In recent years, it has been reported that human multipotential mesenchymal stem cells (hMSCs) differentiate into hepatocyte-like cells. The fetal lung is one of the largest organs containing many MSCs that can be easily obtained. Whether MSCs from fetal lung can differentiate into hepatocytes or bile duct cells is an important issue in basic medicine and clinical application. We isolated fetal lung cells, and expanded and analyzed them. At passage 4, their morphologic, immunophenotyping and cytokine secretions were similar to adult bone marrow-derived MSCs. We conclude that these cells from fetal lung are MSCs, indicating that human fetal lung is an ideal source of hMSCs. hMSCs from fetal lung induced in special differentiation medium showed homogeneous and small polygonal endothelial-like morphology, expressing weak mRNA, as well as Alb and AFP. This implies that hMSCs from fetal lung can differentiate into hepatocyte-like cells.     

Functional studies of mesenchymal stem cells derived from adult human adipose tissue

Recent evidence suggests that cells with the properties of human mesenchymal stem cells (hMSCs) can be derived from adult peripheral tissues, including adipose tissue, muscle and dermis. We isolated hMSCs from the stromal-vascular portion of subcutaneous adipose tissue from seven adult subjects. These cells could be readily differentiated into cells of the chondrocyte, osteocyte and adipocyte lineage demonstrating their multipotency. We studied the functional properties of hMSCs-derived adipocytes and compared them with adipocytes differentiated from hMSCs obtained from bone marrow (BM-hMSC). The two cell types displayed similar lipolytic capacity upon stimulation with catecholamines, including a pronounced antilipolytic effect mediated through alpha2A-adrenoceptors, a typical trait in human but not rodent fat cells. Furthermore, both cell types secreted the fat cell-specific factors leptin and adiponectin in comparable amounts per time unit. The fat tissue-derived hMSCs retained their differentiation capacity up to at least fifteen passages. We conclude that hMSCs derived from adult human adipose tissue can be differentiated into fully functional adipocytes with a similar, if not identical, phenotype as that observed in cells derived from BM-hMSCs. Human adipose-tissue-derived MSCs could therefore constitute an efficient and easily obtainable renewable cellular source for studies of adipocyte biology.     

Prolonged Mechanical Stretch Initiates Intracellular Calcium Oscillations in Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a promising candidate for cell-based therapy in regenerative medicine. These stem cells can interact with their mechanical microenvironment to control their functions. External mechanical cues can be perceived and transmitted into intracellular calcium dynamics to regulate various cellular processes. Recent studies indicate that human MSCs (hMSCs) exhibit a heterogeneous nature with a subset of hMSCs lacking spontaneous calcium oscillations. In this study, we studied whether and how external mechanical tension can be applied to trigger and restore the intracellular calcium oscillation in these hMSCs lacking spontaneous activities. Utilizing the fluorescence resonance energy transfer (FRET) based calcium biosensor, we found that this subpopulation of hMSCs can respond to a prolonged mechanical stretch (PMS). Further results revealed that the triggering of calcium oscillations in these cells is dependent on the calcium influx across the plasma membrane, as well as on both cytoskeletal supports, myosin light chain kinase (MLCK)-driven actomyosin contractility, and phospholipase C (PLC) activity. Thus, our report confirmed that mechanical tension can govern the intracellular calcium oscillation in hMSCs, possibly via the control of the calcium permeability of channels at the plasma membrane. Our results also provide novel mechanistic insights into how hMSCs sense mechanical environment to regulate cellular functions.     

Signalling pathway in the induction of neurite outgrowth in human mesenchymal stem cells

Recent in vivo transplantation studies have shown that mesenchymal stem cells (MSCs) were able to differentiate into mesoderm-derived cell types as well as cells with neuroectodermal characteristics, suggesting that transdifferentiation occurs in the mammalian system. We have reported an immortalized line of human MSCs (hMSCs), KP-hMSCs, which expresses CD29, CD44, CD90, and CD105, and complies with the characteristics shared by mere hMSCs. In a current experiment, we further demonstrated that expanded KP-hMSCs exhibited markers of neuroepithelial or neural precursor cells, such as Nestin, Musashi-1, Vimentin, NCAM, Pax-6, and Sox-9. KP-hMSCs simultaneously expressed proteins of the neuronal, astrocyte, and oligodendrocyte lineages during culture expansion; in addition, they initiated neurite outgrowth and eradicated protein expressions of astrocyte and oligodendrocyte lineages in response to the elevated signaling of the cAMP-PKA pathway after serum depletion in a defined neural induction medium. From the current results, KP-hMSCs may be used to elucidate molecular signaling on the neural differentiation of adult human non-neural tissues. We also presented evidence for the possibility that adult MSCs and fetal neuroepithelial or neural precursor cells both provide for the continual maintenance and repair of the postnatal neural tissues and may derive from the same origin or have one deriving from the other.     

Calcium channels and their role in regenerative medicine

Stem cells hold indefinite self-renewable capability that can be differentiated into all desired cell types.Based on their plasticity potential,they are divided into totipotent(morula stage cells),pluripotent(embryonic stem cells),multipotent(hematopoietic stem cells,multipotent adult progenitor stem cells,and mesenchymal stem cells[MSCs]),and unipotent(progenitor cells that differentiate into a single lineage)cells.Though bone marrow is the primary source of multipotent stem cells in adults,other tissues such as adipose tissues,placenta,amniotic fluid,umbilical cord blood,periodontal ligament,and dental pulp also harbor stem cells that can be used for regenerative therapy.In addition,induced pluripotent stem cells also exhibit fundamental properties of self-renewal and differentiation into specialized cells,and thus could be another source for regenerative medicine.Several diseases including neurodegenerative diseases,cardiovascular diseases,autoimmune diseases,virus infection(also coronavirus disease 2019)have limited success with conventional medicine,and stem cell transplantation is assumed to be the best therapy to treat these disorders.Importantly,MSCs,are by far the best for regenerative medicine due to their limited immune modulation and adequate tissue repair.Moreover,MSCs have the potential to migrate towards the damaged area,which is regulated by various factors and signaling processes.Recent studies have shown that extracellular calcium(Ca²⁺)promotes the proliferation of MSCs,and thus can assist in transplantation therapy.Ca²⁺signaling is a highly adaptable intracellular signal that contains several components such as cell-surface receptors,Ca²⁺channels/pumps/exchangers,Ca²⁺buffers,and Ca²⁺sensors,which together are essential for the appropriate functioning of stem cells and thus modulate their proliferative and regenerative capacity,which will be discussed in this review.     

A link between the accumulation of DNA damage and loss of multi‐potency of human mesenchymal stromal cells

Human mesenchymal stromal cells (hMSCs) represent an attractive cell source for clinic applications. Besides being multi‐potent, recent clinical trials suggest that they secrete both trophic and immunomodulatory factors, allowing allogenic MSCs to be used in a wider variety of clinical situations. The yield of prospective isolation is however very low, making expansion a required step toward clinical applications. Unfortunately, this leads to a significant decrease in their stemness. To identify the mechanism behind loss of multi‐potency, hMSCs were expanded until replicative senescence and the concomitant molecular changes were characterized at regular intervals. We observed that, with time of culture, loss of multi‐potency was associated with both the accumulation of DNA damage and the respective activation of the DNA damage response pathway, suggesting a correlation between both phenomena. Indeed, exposing hMSCs to DNA damage agents led to a significant decrease in the differentiation potential. We also showed that hMSCs are susceptible to accumulate DNA damage upon in vitro expansion, and that although hMSCs maintained an effective nucleotide excision repair activity, there was a progressive accumulation of DNA damage. We propose a model in which DNA damage accumulation contributes to the loss of differentiation potential of hMSCs, which might not only compromise their potential for clinical applications but also contribute to the characteristics of tissue ageing.     

Dkk-1-derived synthetic peptides and lithium chloride for the control and recovery of adult stem cells from bone marrow

It is established that human mesenchymal stem cells (hMSCs) from bone marrow are a source of osteoblast progenitors in vivo and under appropriate conditions, differentiate into osteoblasts ex vivo. Because hMSCs are recovered by iliac crest aspirate and enriched by virtue of their adherence to tissue culture plastic, the cells provide a convenient ex vivo model for the study of osteogenic tissue repair in an experimentally accessible system. Recent advances in the field of skeletal development and osteogenesis have demonstrated that signaling through the canonical wingless (Wnt) pathway is critical for the differentiation of progenitor cell lines into osteoblasts. Inhibition of such signals can predispose MSCs to cell cycle entry and inhibit osteogenesis. Here, we report that synthetic peptides derived from the second cysteine-rich domain of the canonical Wnt inhibitor Dickkopf-1 (Dkk-1) have utility in controlling the growth and recovery of hMSCs from bone marrow stroma. Three peptides corresponding to residues 217-269 in Dkk-1 were each found to enhance the proliferation of hMSCs in culture over 2 days. The most active peptide exhibited agonistic characteristics in that it ablated the proliferation lag observed when cultures of hMSCs receive fresh medium. It also reduced the expression of endogenous Dkk-1 (Gregory, C. A., Singh, H., and Prockop, D. J. (2003) J. Biol. Chem. 278, 28067-28078). When the cytosolic level of beta-catenin was elevated by addition of LiCl to cultures of hMSCs, the peptide also accelerated degradation of beta-catenin on withdrawal of lithium. A second peptide, corresponding to residues 184-204 had preferential and high affinity for hMSCs in the log phase of proliferation. Peptide overlay assays on hMSC lysates confirmed that the peptide bound to a 184-kDa protein corresponding to the molecular mass of LRP6. Cells recovered by this peptide had enhanced osteogenic potential but less chondrogenic potential compared with controls. Because Wnt antagonists increase the number of non-committed hMSCs in culture, they may be of use in increasing the rate of osseous wound healing in vivo by increasing the level of systemically migrating hMSCs. Therefore, such molecules could contribute to the development of a novel family of pharmaceutical agents for the improvement of the healing process in humans.     

Persistent DNA damage‐induced premature senescence alters the functional features of human bone marrow mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well‐known anti‐tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated β‐galactosidase activity and enlarged γH2AX foci co‐localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence‐associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour‐promoting behaviour.     

Growth and functional harvesting of human mesenchymal stromal cells cultured on a microcarrier‐based system

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost‐effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier‐based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM‐MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM‐MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ～4.82 ± 1.18 × 10⁵ cell mL^?1 at day 7, representing a 3.9‐fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP‐compliant large‐scale production system for hMSCs for cellular therapy. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:889–895, 2014     

Human mesenchymal stem cells express neuronal markers after osteogenic and adipogenic differentiation

Mesenchymal stem cells (MSCs) are multipotent cells that are able to differentiate into mesodermal lineages (osteogenic, adipogenic, chondrogenic), but also towards non-mesodermal derivatives (e.g. neural cells). Recent in vitro studies revealed that, in the absence of any kind of differentiation stimuli, undifferentiated MSCs express neural differentiation markers, but the literature data do not all concur. Considering their promising therapeutic potential for neurodegenerative diseases, it is very important to expand our knowledge about this particular biological property of MSCs. In this study, we confirmed the spontaneous expression of neural markers (neuronal, glial and progenitor markers) by undifferentiated human MSCs (hMSCs) and in particular, we demonstrated that the neuronal markers βIII-tubulin and NeuN are expressed by a very high percentage of hMSCs, regardless of the number of culture passages and the culture conditions. Moreover, the neuronal markers βIII-tubulin and NeuN are still expressed by hMSCs after in vitro osteogenic and adipogenic differentiation. On the other hand, chondrogenically differentiated hMSCs are negative for these markers. Our findings suggest that the expression of neuronal markers could be common to a wide range of cellular types and not exclusive for neuronal lineages. Therefore, the expression of neuronal markers alone is not sufficient to demonstrate the differentiation of MSCs towards the neuronal phenotype. Functional properties analysis is also required.     

Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres

Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.     

Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination

Mesenchymal stem/stromal cells (MSCs) can be isolated from most adult tissues and hold considerable promise for tissue regenerative therapies. Some of the potential advantages that MSCs have over other adult stem cell types include: (1) their relative ease of isolation, culture and expansion; (2) their immunomodulatory properties; (3) they can provide trophic support to injured tissues; (4) they can be transduced by retroviral vectors at a high efficiency; (5) they have an ability to home to sites of inflammation and injury. Collectively these characteristics suggest that MSCs are attractive vehicles for cell and gene therapy applications. In the current study, we investigated whether transplantation of human adipose-derived MSCs (Ad-MSCs) engineered to overexpress the anti-inflammatory cytokine interleukin (IL)-4 was efficacious in experimental autoimmune encephalomyelitis (EAE). Ad-MSCs transduced with a bicistronic lentiviral vector encoding mouse IL-4 and enhanced green fluorescent protein (Ad-IL4-MSCs) stably expressed, relatively high levels of both transgenes. Importantly the phenotypic and functional attributes of Ad-IL4-MSCs, such as the expression of homing molecules and differentiation capacity, was not altered by the transduction process. Notably, the early administration of Ad-IL4-MSCs in mice with EAE at the time of T-cell priming attenuated clinical disease. This protective effect was associated with a reduction in peripheral MOG-specific T-cell responses and a shift from a pro- to an anti-inflammatory cytokine response. These data suggest that the delivery of Ad-MSCs genetically engineered to express anti-inflammatory cytokines may provide a rational approach to promote immunomodulation and tissue protection in a number of inflammatory and degenerative diseases including multiple sclerosis.     

Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination

Mesenchymal stem/stromal cells (MSCs) can be isolated from most adult tissues and hold considerable promise for tissue regenerative therapies. Some of the potential advantages that MSCs have over other adult stem cell types include: (1) their relative ease of isolation, culture and expansion; (2) their immunomodulatory properties; (3) they can provide trophic support to injured tissues; (4) they can be transduced by retroviral vectors at a high efficiency; (5) they have an ability to home to sites of inflammation and injury. Collectively these characteristics suggest that MSCs are attractive vehicles for cell and gene therapy applications. In the current study, we investigated whether transplantation of human adipose-derived MSCs (Ad-MSCs) engineered to overexpress the anti-inflammatory cytokine interleukin (IL)-4 was efficacious in experimental autoimmune encephalomyelitis (EAE). Ad-MSCs transduced with a bicistronic lentiviral vector encoding mouse IL-4 and enhanced green fluorescent protein (Ad-IL4-MSCs) stably expressed, relatively high levels of both transgenes. Importantly the phenotypic and functional attributes of Ad-IL4-MSCs, such as the expression of homing molecules and differentiation capacity, was not altered by the transduction process. Notably, the early administration of Ad-IL4-MSCs in mice with EAE at the time of T-cell priming attenuated clinical disease. This protective effect was associated with a reduction in peripheral MOG-specific T-cell responses and a shift from a pro- to an anti-inflammatory cytokine response. These data suggest that the delivery of Ad-MSCs genetically engineered to express anti-inflammatory cytokines may provide a rational approach to promote immunomodulation and tissue protection in a number of inflammatory and degenerative diseases including multiple sclerosis.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

Collagen type XV and the ‘osteogenic status’

We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different osteogenic potential and by osteoblasts (hOBs) that are able to grow in culture medium with or without calcium. Analysing the osteogenic process, we have shown that ColXV basal levels are lower in cells less prone to osteo‐induction such as hMSCs from Wharton Jelly (hWJMSCs), compared to hMSCs that are prone to osteo‐induction such as those from the bone marrow (hBMMSCs). In the group of samples identified as ‘mineralized MSCs’, during successful osteogenic induction, ColXV protein continued to be detected at substantial levels until early stage of differentiation, but it significantly decreased and then disappeared at the end of culture when the matrix formed was completely calcified. The possibility to grow hOBs in culture medium without calcium corroborated the results obtained with hMSCs demonstrating that calcium deposits organized in a calcified matrix, and not calcium ‘per se’, negatively affected ColXV expression. As a whole, our data suggest that ColXV may participate in ECM organization in the early‐phases of the osteogenic process and that this is a prerequisite to promote the subsequent deposition of mineral matrix.