Human Periosteum-derived Progenitor Cells Research Articles

In vitro and in vivo evaluation of periosteum-derived cells and iPSC-derived chondrocytes encapsulated in GelMA for osteochondral tissue engineering.

Osteochondral defects are deep joint surface lesions that affect the articular cartilage and the underlying subchondral bone. In the current study, a tissue engineering approach encompassing individual cells encapsulated in a biocompatible hydrogel is explored in vitro and in vivo. Cell-laden hydrogels containing either human periosteum-derived progenitor cells (PDCs) or human induced pluripotent stem cell (iPSC)-derived chondrocytes encapsulated in gelatin methacryloyl (GelMA) were evaluated for their potential to regenerate the subchondral mineralized bone and the articular cartilage on the joint surface, respectively. PDCs are easily isolated and expanded progenitor cells that are capable of generating mineralized cartilage and bone tissue in vivo via endochondral ossification. iPSC-derived chondrocytes are an unlimited source of stable and highly metabolically active chondrocytes. Cell-laden hydrogel constructs were cultured for up to 28 days in a serum-free chemically defined chondrogenic medium. On day 1 and day 21 of the differentiation period, the cell-laden constructs were implanted subcutaneously in nude mice to evaluate ectopic tissue formation 4 weeks post-implantation. Taken together, the data suggest that iPSC-derived chondrocytes encapsulated in GelMA can generate hyaline cartilage-like tissue constructs with different levels of maturity, while using periosteum-derived cells in the same construct type generates mineralized tissue and cortical bone in vivo. Therefore, the aforementioned cell-laden hydrogels can be an important part of a multi-component strategy for the manufacturing of an osteochondral implant.

Identifying Fibroblast Growth Factor Receptor 3 as a Mediator of Periosteal Osteochondral Differentiation through the Construction of microRNA-Based Interaction Networks.

Human periosteum-derived progenitor cells (hPDCs) have the ability to differentiate towards both the chondrogenic and osteogenic lineages. This coordinated and complex osteochondrogenic differentiation process permits endochondral ossification and is essential in bone development and repair. We have previously shown that humanised cultures of hPDCs enhance their osteochondrogenic potentials in vitro and in vivo; however, the underlying mechanisms are largely unknown. This study aimed to identify novel regulators of hPDC osteochondrogenic differentiation through the construction of miRNA-mRNA regulatory networks derived from hPDCs cultured in human serum or foetal bovine serum as an alternative in silico strategy to serum characterisation. Sixteen differentially expressed miRNAs (DEMis) were identified in the humanised culture. In silico analysis of the DEMis with TargetScan allowed for the identification of 1503 potential miRNA target genes. Upon comparison with a paired RNAseq dataset, a 4.5% overlap was observed (122 genes). A protein-protein interaction network created with STRING interestingly identified FGFR3 as a key network node, which was further predicted using multiple pathway analyses. Functional analysis revealed that hPDCs with the activating mutation FGFR3N540K displayed increased expressions of chondrogenic gene markers when cultured under chondrogenic conditions in vitro and displayed enhanced endochondral bone formation in vivo. A further histological analysis uncovered known downstream mediators involved in FGFR3 signalling and endochondral ossification to be upregulated in hPDC FGFR3N540K-seeded implants. This combinational approach of miRNA-mRNA-protein network analysis with in vitro and in vivo characterisation has permitted the identification of FGFR3 as a novel mediator of hPDC biology. Furthermore, this miRNA-based workflow may also allow for the identification of drug targets, which may be of relevance in instances of delayed fracture repair.

Real-Time Cell Growth Control Using a Lactate-Based Model Predictive Controller

Providing a cost-efficient feeding strategy for cell expansion processes remains a challenging task due to, among other factors, donor variability. The current method to use a fixed medium replacement strategy for all cell batches results often in either over- or underfeeding these cells. In order to take into account the individual needs of the cells, a model predictive controller was developed in this work. Reference experiments were performed by expanding human periosteum derived progenitor cells (hPDCs) in tissue flasks to acquire reference data. With these data, a time-variant prediction model was identified to describe the relation between the accumulated medium replaced as the control input and the accumulated lactate produced as the process output. Several forecast methods to predict the cell growth process were designed using multiple collected datasets by applying transfer function models or machine learning. The first controller experiment was performed using the accumulated lactate values from the reference experiment as a static target function over time, resulting in over- or underfeeding the cells. The second controller experiment used a time-adaptive target function by combining reference data as well as current measured real-time data, without over- or underfeeding the cells.

Microcarrier Screening and Evaluation for Dynamic Expansion of Human Periosteum-Derived Progenitor Cells in a Xenogeneic Free Medium.

An increasing need toward a more efficient expansion of adherent progenitor cell types arises with the advancements of cell therapy. The use of a dynamic expansion instead of a static planar expansion could be one way to tackle the challenges of expanding adherent cells at a large scale. Microcarriers are often reported as a biomaterial for culturing cells in suspension. However, the type of microcarrier has an effect on the cell expansion. In order to find an efficient expansion process for a specific adherent progenitor cell type, it is important to investigate the effect of the type of microcarrier on the cell expansion. Human periosteum-derived progenitor cells are extensively used in skeletal tissue engineering for the regeneration of bone defects. Therefore, we evaluated the use of different microcarriers on human periosteum-derived progenitor cells. In order to assess the potency, identity and viability of these cells after being cultured in the spinner flasks, this study performed several in vitro and in vivo analyses. The novelty of this work lies in the combination of screening different microcarriers for human periosteum-derived progenitor cells with in vivo assessments of the cells’ potency using the microcarrier that was selected as the most promising one. The results showed that expanding human periosteum-derived progenitor cells in spinner flasks using xeno-free medium and Star-Plus microcarriers, does not affect the potency, identity or viability of the cells. The potency of the cells was assured with an in vivo evaluation, where bone formation was achieved. In summary, this expansion method has the potential to be used for large scale cell expansion with clinical relevance.

MicroRNA Profiling in Mesenchymal Stromal Cells: the Tissue Source as the Missing Piece in the Puzzle of Ageing.

Ageing is among the main risk factors for human disease onset and the identification of the hallmarks of senescence remains a challenge for the development of appropriate therapeutic target in the elderly. Here, we compare senescence-related changes in two cell populations of mesenchymal stromal cells by analysing their miRNA profiling: Human Dental Pulp Stromal Cells (hDPSCs) and human Periosteum-Derived Progenitor Cells (hPDPCs). After these cells were harvested, total RNA extraction and whole genome miRNA profiling was performed, and DIANA-miRPath analysis was applied to find the target/pathways. Only 69 microRNAs showed a significant differential expression between dental pulp and periosteum progenitor cells. Among these, 24 were up regulated, and 45 were downregulated in hDPSCs compared to hPDPCs. Our attention was centered on miRNAs (22 upregulated and 34 downregulated) involved in common pathways for cell senescence (i.e. p53, mTOR pathways), autophagy (i.e. mTOR and MAPK pathways) and cell cycle (i.e. MAPK pathway). The p53, mTOR and MAPK signaling pathways comprised 43, 37 and 112 genes targeted by all selected miRNAs, respectively. Our finding is consistent with the idea that the embryological origin influences cell behavior and the ageing process. Our study strengthens the hypothesis that ageing is driven by numerous mediators interacting through an intricate molecular network, which affects adult stem cells self-renewal capability. Graphical abstract.

Developmental engineering of living implants for deep osteochondral joint surface defects.

The repair of deep osteochondral joint surface defects represents a significant unmet clinical need. Importantly, untreated lesions lead to a high rate of osteoarthritis. The current strategies to repair these defects include osteochondral autograft transplantation or "sandwich" strategies combining bone autografts with autologous chondrocyte implantation, with poorly documented long-term outcomes. In this study, we first investigated the capacity of juvenile osteochondral grafts (OCGs) to repair osteochondral defects in skeletally mature rats. With this regenerative model in view, we produced a new biological, bilayered and scaffold-free Tissue Engineered construct (bTEC) for the repair of a deep osteochondral defect of the rat knee. Cylindrical OCGs were excised from the femoral intercondylar groove of the knee of skeletally immature rats (5weeks) and transplanted into osteochondral defects created in skeletally mature rats (11weeks). To create bTECs, micromasses (μMasses) of human periosteum-derived progenitor cells (hPDCs) and human articular chondrocytes (hACs) were produced in vitro using previously optimized chemically defined medium formulations containing growth and differentiation factors including bone morphogenetic proteins. These two μMass types were subsequently implanted as bilayered constructs into osteochondral defects in nude rats. At 4 and 16weeks after surgery, the knees were collected and processed for subsequent 3D imaging analysis and histological evaluation. Micro-computed tomography (μCT), H&E, and Safranin O staining were used to evaluate the degree and quality of tissue repair. The osteochondral unit of the knee joint in 5weeks old rats exhibits an immature phenotype, displaying active subchondral bone formation through endochondral ossification and the absence of a tidemark. When transplanted into skeletally mature animals, the immature OCGs resumed their maturation process, i.e., formed new subchondral bone, established the tidemark, and maintained their Safranin O-positive hyaline cartilage at 16weeks after transplantation. The bTECs (hPDCs + hACs) could partially recapitulate the biology as seen with the immature OCGs, including the formation of the joint surface architecture with typical zonation, ranging from non-mineralized hyaline cartilage in the superficial layers to a progressively mineralized matrix at the interface with a new subchondral bone plate. Cell-based TE constructs mimicking immature OCGs and displaying a hierarchically organized structure comprising of different tissue forming units seem an attractive strategy to treat deep osteochondral defects of the knee.

Deciphering the combined effect of bone morphogenetic protein 6 and calcium phosphate on bone formation capacity of periosteum derived cell-based tissue engineering constructs

Cell based combination products with growth factors on optimal carriers represent a promising tissue engineering strategy to treat large bone defects. In this concept, bone morphogenetic protein (BMP) and calcium phosphate (CaP)-based scaffolds can act as potent components of the constructs to steer stem cell specification, differentiation and initiate subsequent in vivo bone formation. However, limited insight into BMP dosage and the cross-talk between BMP and CaP materials, hampers the optimization of in vivo bone formation and subsequent clinical translation. Herein, we combined human periosteum derived progenitor cells with different doses of BMP6 and with three types of clinical grade CaP-scaffolds (ChronOs®, ReproBone™, & CopiOs®). Comprehensive cellular and molecular analysis was performed based on in vitro cell metabolic activity and signaling pathway activation, as well as in vivo ectopic bone forming capacity after 2 weeks and 5 weeks in nude mice. Our data showed that cells seeded on CaP scaffolds with an intermediate Ca2+ release rate combined with low or medium dosage of BMP6 demonstrated a robust new bone formation after 5 weeks, which was contributed by both donor and host cells. This phenomenon might be due to the delicate balance between Ca2+ and BMP pathways, allowing an appropriate activation of the canonical BMP signaling pathway that is required for in vivo bone formation. For high BMP6 dosage, we found that the BMP6 dosage overrides the effect of the Ca2+ release rate and this appeared to be a dominant factor for ectopic bone formation. Taken together, this study illustrates the importance of matching BMP dosage and CaP properties to allow an appropriate activation of canonical BMP signaling that is crucial for in vivo bone formation. It also provides insightful knowledge with regard to clinical translation of cell-based constructs for bone regeneration. Statement of SignificanceThe combination of bone morphogenetic proteins (BMP) and calcium phosphate (CaP)-based biomaterials with mesenchymal stromal cells represents a promising therapeutic strategy to treat large bone defects, an unmet medical need. However, there is limited insight into the optimization of these combination products, which hampers subsequent successful clinical translation. Our data reveal a delicate balance between Ca2+ and BMP pathways, allowing an appropriate activation of canonical BMP signaling required for in vivo bone formation. Our findings illustrate the importance of matching BMP dosage and CaP properties in the development of cell-based constructs for bone regeneration.

Serum of patients with active rheumatoid arthritis inhibits differentiation of osteochondrogenic precursor cells

Delayed fracture healing is frequently experienced in patients with systemic inflammation such as during rheumatoid arthritis (RA). The reasons for this are diverse, but could also be caused by inflammatory cytokines and/or growth factors in serum from patients with active disease. We hypothesized that serum from patients with active RA contains circulating inflammatory factors that inhibit differentiation of osteochondrogenic precursors. Serum was obtained from 15 patients with active RA (active RA-sera) and from the same patients in clinical remission 1 year later (remission RA-sera; controls). The effect of active RA-sera on osteochondrogenic differentiation of chondrogenic ATDC5 cells and primary human periosteum-derived progenitor cells (HPDC) was determined in micromass culture. In ATDC5 cells, active RA-sera reduced Ki67 transcription levels by 40% and cartilage matrix accumulation by 14% at day 14, and Alp transcription levels by 16%, and matrix mineralization by 17% at day 21 compared with remission RA-sera. In HPDCs, active RA-sera inhibited metabolic activity by 8%, SOX9 transcription levels by 14%, and cartilage matrix accumulation by 7% at day 7 compared with remission RA-sera. In conclusion, sera from patients with active RA negatively affect differentiation of osteochondrogenic precursors, and as a consequence may contribute to delayed fracture healing in these patients.

Serum of patients with active rheumatoid arthritis inhibits differentiation of osteochondrogenic precursor cells

ABSTRACTDelayed fracture healing is frequently experienced in patients with systemic inflammation such as during rheumatoid arthritis (RA). The reasons for this are diverse, but could also be caused by inflammatory cytokines and/or growth factors in serum from patients with active disease. We hypothesized that serum from patients with active RA contains circulating inflammatory factors that inhibit differentiation of osteochondrogenic precursors. Serum was obtained from 15 patients with active RA (active RA-sera) and from the same patients in clinical remission 1 year later (remission RA-sera; controls). The effect of active RA-sera on osteochondrogenic differentiation of chondrogenic ATDC5 cells and primary human periosteum–derived progenitor cells (HPDC) was determined in micromass culture. In ATDC5 cells, active RA-sera reduced Ki67 transcription levels by 40% and cartilage matrix accumulation by 14% at day 14, and Alp transcription levels by 16%, and matrix mineralization by 17% at day 21 compared with remission RA-sera. In HPDCs, active RA-sera inhibited metabolic activity by 8%, SOX9 transcription levels by 14%, and cartilage matrix accumulation by 7% at day 7 compared with remission RA-sera. In conclusion, sera from patients with active RA negatively affect differentiation of osteochondrogenic precursors, and as a consequence may contribute to delayed fracture healing in these patients.

Early BMP, Wnt and Ca2+/PKC pathway activation predicts the bone forming capacity of periosteal cells in combination with calcium phosphates

The development of osteoinductive calcium phosphate- (CaP) based biomaterials has, and continues to be, a major focus in the field of bone tissue engineering. However, limited insight into the spatiotemporal activation of signalling pathways has hampered the optimisation of in vivo bone formation and subsequent clinical translation. To gain further knowledge regarding the early molecular events governing bone tissue formation, we combined human periosteum derived progenitor cells with three types of clinically used CaP-scaffolds, to obtain constructs with a distinct range of bone forming capacity in vivo. Protein phosphorylation together with gene expression for key ligands and target genes were investigated 24 hours after cell seeding in vitro, and 3 and 12 days post ectopic implantation in nude mice. A computational modelling approach was used to deduce critical factors for bone formation 8 weeks post implantation. The combined Ca2+-mediated activation of BMP-, Wnt- and PKC signalling pathways 3 days post implantation were able to discriminate the bone forming from the non-bone forming constructs. Subsequently, a mathematical model able to predict in vivo bone formation with 96% accuracy was developed. This study illustrates the importance of defining and understanding CaP-activated signalling pathways that are required and sufficient for in vivo bone formation. Furthermore, we demonstrate the reliability of mathematical modelling as a tool to analyse and deduce key factors within an empirical data set and highlight its relevance to the translation of regenerative medicine strategies.

Transplantation of insulin-producing cells differentiated from human periosteum-derived progenitor cells ameliorate hyperglycemia in diabetic mice.

Periosteum-derived progenitor cells (PDPCs) isolated from the adult periosteum can differentiate into several specific cell types. In this study, we examined the characteristics of human PDPCs and insulin-producing cells (IPCs) differentiated from PDPCs and their ability to ameliorate hyperglycemia when transplanted into streptozotocin-induced nonobese diabetic-severe combined immunodeficiency diabetic mice. Periosteum-derived progenitor cells were isolated from patients, expanded in culture, and subjected to a three-step differentiation protocol to produce IPCs. The expression of immunogenic, pluripotent, and pancreatic markers was examined, and glucose-stimulated insulin release in vitro was also assessed. Insulin-producing cells that differentiated from PDPCs were transplanted under the kidney capsule of streptozotocin-induced diabetic mice, and glucose levels and glucose tolerance were measured. We found that PDPCs expressed the mesenchymal stem cell markers CD73, CD90, and CD105 and the pluripotent markers, octamer-binding transcription factor 4 and Nanog, but not sex-determining region Y-box 2 or Rex1. Periosteum-derived progenitor cells expressed human leukocyte antigen-ABC but did not express human leukocyte antigen-DR or the costimulatory molecules CD80 and CD86. Differentiated IPCs expressed pancreatic hormones (insulin, glucagon, somatostatin, and glucose transporter 2), hormone processing, and secretion molecules (prohormone convertase-1 and convertase-2, Kir6.2), and pancreatic transcription factors (neurogenin 3, pancreatic and duodenal homeobox 1, sex-determining region Y-box 17). When IPCs were stimulated with glucose in vitro, insulin secretion was elevated. Transplantation of IPCs under the kidney capsules of diabetic mice improved hyperglycemia and glucose tolerance. Human insulin was detected in the serum and kidney sections of mice transplanted with IPCs differentiated from PDPCs. These results suggest that IPCs differentiated from PDPCs might be an alternative source of β cells for treating diabetes.

In vitro cardiomyogenic potential of human periosteum-derived progenitor cells

In vitro cardiomyogenic potential of human periosteum-derived progenitor cells

Human periosteum-derived progenitor cells express distinct chemokine receptors and migrate upon stimulation with CCL2, CCL25, CXCL8, CXCL12, and CXCL13

For bone repair, transplantation of periosteal progenitor cells (PCs), which had been amplified within supportive scaffolds, is applied clinically. More innovative bone tissue engineering approaches focus on the in situ recruitment of stem and progenitor cells to defective sites and their subsequent use for guided tissue repair. Chemokines are known to induce the directed migration of bone marrow CD34 − mesenchymal stem cells (MSCs). The aim of our study was to determine the chemokine receptor expression profile of human CD34 − PCs and to demonstrate that these cells migrate upon stimulation with selected chemokines. PCs were isolated from periosteum of the mastoid bone and displayed a homogenous cell population presenting an MSC-related cell-surface antigen profile (ALCAM +, SH2 +, SH3 +, CD14 −, CD34 −, CD44 +, CD45 −, CD90 +). The expression profile of chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that PCs express receptors of all four chemokine subfamilies CC, CXC, CX 3C, and C. Migration of PCs and a dose-dependent migratory effect of the chemokines CCL2 (MCP1), CCL25 (TECK), CXCL8 (IL8), CXCL12 (SDF1α), and CXCL13 (BCA1), but not CCL22 (MDC) were demonstrated using a 96-multiwell chemotaxis assay. In conclusion, for the first time, here we report that human PCs express chemokine receptors, present their profile, and demonstrate a dose-dependent migratory effect of distinct chemokines on these cells. These results are promising towards in situ bone repair therapies based on guiding PCs to bone defects, and encourage further in vivo studies.

Chondrogenesis of human periosteum-derived progenitor cells in atelocollagen

Periosteum-derived progenitor cells (PDPCs) could be differentiated into cartilage using atelocollagen as a carrier and in the presence of transforming growth factor-beta3 (TGF-beta3). Chondrogenesis was verified by RT-PCR and Western blotting. Expression of the type II collagen mRNA was found from the differentiated PDPCs in atelocollagen 3 weeks after chondrogenic induction. The chondrogenic potential of the PDPCs was also verified by histochemical staining for type II collagen protein. Increased production of glycosaminoglycan shows that the PDPCs in atelocollagen could differentiate into chondrocytes under a chondrogenic environment. PDPCs can therefore be used as a cell source for cell-based therapies targeted toward the articular cartilage of the knee.

Osteogenic potential of human periosteum-derived progenitor cells in PLGA scaffold using allogeneic serum

The use of periosteum-derived progenitor cells (PCs) combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional (3D) poly-lactic-co-glycolic acid (PLGA) fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type I collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules of neo-bone tissue formation and the presence of matrix mineralization were confirmed by positive von Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.

Isolation of human periosteum-derived progenitor cells using immunophenotypes for chondrogenesis

Periosteum-derived progenitor cells (PDPCs) were isolated by characteristic surface markers. Reproducibility of immunophenotypes of the PDPCs was characterized by flow cytometric analysis using fluorescence-activated cell sorter (FACS). SH2(+), SH3(+), SH4(+), CD9(+), CD90(+) and CD105(+) were important eternal characteristic cell surface markers for the PDPCs. The characterized PDPCs maintained their chondrogenic potential in pellet cultures until the 15th passage from primary cell culture.