Mittal Shah | University College London (original) (raw)

Papers by Mittal Shah

Tissue Engineering Part A, Sep 1, 2018

Complications that arise from impaired fracture healing have considerable socioeconomic implicati... more Complications that arise from impaired fracture healing have considerable socioeconomic implications. Current research in the field of bone tissue engineering predominantly aims to mimic the mature bone tissue microenvironment. This approach, however, may produce implants that are intrinsically unresponsive to the cues present during the initiation of fracture repair. As such, this study describes the development of decellularised xenogeneic hyaline cartilage matrix in an attempt to mimic the initial reparative phase of fracture repair. Three approaches based on vacuum-assisted osmotic shock (Vac-OS), Triton X (Vac-Stx) and SDS (Vac-SDS) were investigated. The Vac-OS methodology reduced DNA content below 50ng/mg of tissue, whilst retaining 85% of the sGAG content and as such was selected as the optimal methodology for decellularisation. The resultant Vac-OS scaffolds (dcECM) were also devoid of the immunogenic alpha-gal epitope. Furthermore, minimal disruption to the structural integrity of the dcECM was demonstrated using differential scanning calorimetry (DSC) and fluorescence lifetime imaging microscopy (FLIM). The biological integrity of the dcECM was confirmed by its ability to drive the chondrogenic commitment and differentiation of human chondrocytes and periosteumderived cells respectively. Furthermore, histological examination of dcECM constructs implanted in immunocompetent mice revealed a predominantly M2-macrophage driven regenerative response both at 2 and 8 weeks post-implantation. These findings contrasted with the implanted native costal cartilage that elicited a predominantly M1-macrophage mediated inflammatory response. This study highlights the capacity of dcECM from the Vac-OS methodology to direct the key biological processes of endochondral ossification, thus potentially recapitulating the callus phase of fracture repair.

JBMR Plus

The identity of the cells that form the periosteum during development is controversial with curre... more The identity of the cells that form the periosteum during development is controversial with current dogma suggesting these are derived from a Sox9-positive progenitor. Herein, we characterize a newly created Prrx1eGFP reporter transgenic mouse line during limb formation and postnatally. Interestingly, in the embryo Prrx1eGFP-labeled cells become restricted around the Sox9-positive cartilage anlage without themselves becoming Sox9-positive. In the adult, the Prrx1eGFP transgene live labels a subpopulation of cells within the periosteum that are enriched at specific sites, and this population is diminished in aged mice. The green fluorescent protein (GFP)-labeled subpopulation can be isolated using fluorescence-activated cell sorting (FACS) and represents approximately 8% of all isolated periosteal cells. The GFP-labeled subpopulation is significantly more osteogenic than unlabeled, GFP-negative periosteal cells. In addition, the osteogenic and chondrogenic capacity of periosteal cells in vitro can be extended with the addition of fibroblast growth factor (FGF) to the expansion media. We provide evidence to suggest that osteoblasts contributing to cortical bone formation in the embryo originate from Prrx1eGFP-positive cells within the perichondrium, which possibly piggyback on invading vascular cells and secrete new bone matrix. In summary, the Prrx1eGFP mouse is a powerful tool to visualize and isolate periosteal cells and to quantify their properties in the embryo and adult.

Journal of Clinical Oncology

e19505 Background: Receptor tyrosine kinase-like Orphan Receptor-1 (ROR1) is widely expressed on ... more e19505 Background: Receptor tyrosine kinase-like Orphan Receptor-1 (ROR1) is widely expressed on hematological and solid tumors. NVG-111, a first in class humanized tandem scFv ROR1xCD3 bispecific antibody elicits potent killing of ROR1+ tumor cells in vitro and in vivo. This bispecific T-cell engager (TCE) is being evaluated in a first in human, Phase I trial in patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The predicted therapeutic dose and steady state serum concentrations of NVG-111 were estimated by allometric scaling using relevant doses from a murine PK study. To assess free drug levels in patients following 21 days of continuous infusion of NVG-111, a bespoke, sensitive pharmacokinetics (PK) assay with high levels of specificity and sensitivity was developed. Methods: Anti-idiotype (anti-ID) antibodies directed to anti-ROR1 (αROR1-ID) and anti-CD3 (αCD3-ID) were generated by mouse immunization or by phage display from cu...

International Journal of Experimental Pathology, 2015

Journal of Endocrinology, 2012

AMP-activated protein kinase (AMPK) is a key regulator of cellular and body energy homeostasis. W... more AMP-activated protein kinase (AMPK) is a key regulator of cellular and body energy homeostasis. We previously demonstrated that AMPK activation in osteoblasts increases in vitro bone formation while deletion of the Ampkα1 (Prkaa1) subunit, the dominant catalytic subunit expressed in bone, leads to decreased bone mass in vivo. To investigate the cause of low bone mass in the Ampkα1−/− mice, we analysed bone formation and resorption in the tibia of these mice by dynamic histomorphometry and determined whether bone turnover can be stimulated in the absence of the Ampkα1 subunit. We subjected 12-week-old Ampkα1+/+ and Ampkα1−/− mice to ovariectomy (OVX), intermittent PTH (iPTH) administration (80 μg/kg per day, 5 days/week) or both OVX and iPTH hormonal challenges. Tibiae were harvested from these mice and bone micro-architecture was determined by micro-computed tomography. We show for the first time that Ampkα1−/− mice have a high bone turnover at the basal level in favour of bone reso...

Acta Biomaterialia, 2019

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service... more This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Osteomimetic matrix components alter cell migration and drug response in a 3D tumourengineered osteosarcoma model.

Bioengineering

The control of cell behaviour in an effort to create highly homogeneous cultures is becoming an a... more The control of cell behaviour in an effort to create highly homogeneous cultures is becoming an area of intense research, both to elucidate fundamental biology and for regenerative applications. The extracellular matrix (ECM) controls many cellular processes in vivo, and as such is a rich source of cues that may be translated in vitro. Herein, we describe the creation of cell culture coatings from porcine decellularised hyaline cartilage through enzymatic digestion. Surprisingly, heat-mediated sterilisation created a coating with the capacity to rapidly and robustly induce chondrogenic differentiation of human periosteal cells. This differentiation was validated through the alteration of cell phenotype from a fibroblastic to a cuboidal/cobblestone chondrocyte-like appearance. Moreover, chondrogenic gene expression further supported this observation, where cells cultured on heat sterilised ECM-coated plastic displayed higher expression of COL2A1, ACAN and PRG4 (p < 0.05) compared ...

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

smokeinduced induction of antioxidant enzyme activities in airway leukocytes is absent in active ... more smokeinduced induction of antioxidant enzyme activities in airway leukocytes is absent in active smokers with COPD

Journal of Clinical Oncology

7535 Background: NVG-111 is a first in class, humanized, tandem scFv ROR1xCD3 bispecific T cell e... more 7535 Background: NVG-111 is a first in class, humanized, tandem scFv ROR1xCD3 bispecific T cell engager that mediates potent killing of ROR1+ tumours by engaging an epitope on the Frizzled domain of ROR1 and redirecting T cell activity via the CD3 binder. Methods: This phase I/II study is evaluating NVG-111 in patients with relapsed/refractory (R/R) CLL and MCL who have received ≥2 prior systemic therapies and have achieved a stable, or partial response to the last line of therapy. NVG-111 was delivered as continuous intravenous (cIV) infusion over 21 days per cycle, with each patient typically receiving 3 cycles of treatment. The first 3 single patient cohorts were subjected to accelerated dose titration (ADT) over a range of 0.3-30µg/day. Dose-escalation steps in the subsequent, multi-patient, cohorts were determined using a continuous reassessment method with overdose control. The primary endpoints are safety and determination of MTD/RP2D. Secondary objectives are pharmacokinetic...

Journal of Orthopaedic Translation, 2021

Background: Sclerosteosis, a severe autosomal recessive sclerosing skeletal dysplasia characteris... more Background: Sclerosteosis, a severe autosomal recessive sclerosing skeletal dysplasia characterised by excessive bone formation, is caused by absence of sclerostin, a negative regulator of bone formation that binds LRP5/6 Wnt co-receptors. Current treatment is limited to surgical management of symptoms arising from bone overgrowth. This study investigated the effectiveness of sclerostin replacement therapy in a mouse model of sclerosteosis. Methods: Recombinant wild type mouse sclerostin (mScl) and novel mScl fusion proteins containing a C-terminal human Fc (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), were produced and purified using mammalian expression and standard chromatography methods. In vitro functionality and efficacy of the recombinant proteins were evaluated using three independent biophysical techniques and an in vitro bone nodule formation assay. Pharmacokinetic properties of the proteins were investigated in vivo following a single administration to young female wild type (WT) or SOST knock out (SOST-/-) mice. In a six week proof-of-concept in vivo study, young female WT or SOST-/mice were treated with 10 mg/kg mScl hFc or mScl hFc PD (weekly), or 4.4 mg/kg mScl (daily). The effect of recombinant sclerostin on femoral cortical and trabecular bone parameters were assessed by micro computed tomography (μCT). Results: Recombinant mScl proteins bound to the extracellular domain of the Wnt co-receptor LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in vitro. Pharmacokinetic assessment following a single dose administered to WT or SOST-/mice indicated the presence of hFc increased protein half-life from less than 5 min to at least 1.5 days. Treatment with mScl hFc PD over a six week period resulted in modest but significant reductions in trabecular volumetric bone mineral density (vBMD) and bone volume fraction (BV/TV), of 20% and 15%, respectively. Conclusion: Administration of recombinant mScl hFc PD partially corrected the high bone mass phenotype in SOST-/mice, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/mouse model of sclerosteosis. The translational potential of this article: These findings support the concept that exogenous sclerostin can reduce bone mass, however the modest efficacy suggests that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored.

RMD Open, 2020

ObjectivesInterleukin (IL)-17 signalling has been shown to be a key regulator of disease in ankyl... more ObjectivesInterleukin (IL)-17 signalling has been shown to be a key regulator of disease in ankylosing spondylitis (AS) with several IL-17 blockers currently clinically approved. Despite this, the role of IL-17 in bone pathology is poorly understood. This study aimed to investigate IL-17 signalling in the context of pathological bone formation.MethodsA biomimetic human periosteum-derived cell (hPDC) model of osteogenic differentiation was used in combination with recombinant IL-17 cytokines, T-cell supernatants or serum from patients with AS. IL-17A, IL-17F and bimekizumab monoclonal antibodies were used to block IL-17 cytokine action.ResultsRecombinant IL-17A and IL-17F are pro-osteogenic with respect to hPDC differentiation. T helper 17 or γδ-T cell supernatants also potently stimulated in vitro bone formation, which was blocked deeper by dual inhibition of IL-17A and IL-17F than by neutralisation of IL-17A or IL-17F individually. Osteogenic blockade may be due to an increase in e...

Cytotherapy, 2020

Background: The periosteum is a highly vascularized, collagen-rich tissue that plays a crucial ro... more Background: The periosteum is a highly vascularized, collagen-rich tissue that plays a crucial role in directing bone repair. This is orchestrated primarily by its resident progenitor cell population. Indeed, preservation of periosteum integrity is critical for bone healing. Cells extracted from the periosteum retain their osteochondrogenic properties and as such are a promising basis for tissue engineering strategies for the repair of bone defects. However, the culture expansion conditions and the way in which the cells are reintroduced to the defect site are critical aspects of successful translation. Indeed, expansion in human serum and implantation on biomimetic materials has previously been shown to improve in vivo bone formation. Aim: This study aimed to develop a protocol to allow for the expansion of human periosteum derived cells (hPDCs) in a biomimetic periosteal-like environment. Methods: The expansion conditions were defined through the investigation of the bioactive cues involved in augmenting hPDC proliferative and multipotency characteristics, based on transcriptomic analysis of cells cultured in human serum. Results: Master regulators of transcriptional networks were identified, and an optimized periosteum-derived growth factor cocktail (PD-GFC; containing b-estradiol, FGF2, TNFa, TGFb, IGF-1 and PDGF-BB) was generated. Expansion of hPDCs in PD-GFC resulted in serum mimicry with regard to the cell morphology, proliferative capacity and chondrogenic differentiation. When incorporated into a three-dimensional collagen type 1 matrix and cultured in PD-GFC, the hPDCs migrated to the surface that represented the matrix topography of the periosteum cambium layer. Furthermore, gene expression analysis revealed a down-regulated WNT and TGFb signature and an up-regulation of CREB, which may indicate the hPDCs are recreating their progenitor cell signature. Conclusion: This study highlights the first stage in the development of a biomimetic periosteum, which may have applications in bone repair.

Development, 2019

In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which ... more In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which is characterised, in part, by craniofacial abnormalities. Here, we analyse the role of Dchs1-Fat4 signalling during osteoblast differentiation in mouse. We show that Fat4 and Dchs1 mutants mimic the craniofacial phenotype of the human syndrome and that Dchs1-Fat4 signalling is essential for osteoblast differentiation. In Dchs1/Fat4 mutants, proliferation of osteoprogenitors is increased and osteoblast differentiation is delayed. We show that loss of Dchs1-Fat4 signalling is linked to increased Yap-Tead activity and that Yap is expressed and required for proliferation in osteoprogenitors. In contrast, Taz is expressed in more-committed Runx2-expressing osteoblasts, Taz does not regulate osteoblast proliferation and Taz-Tead activity is unaffected in Dchs1/Fat4 mutants. Finally, we show that Yap and Taz differentially regulate the transcriptional activity of Runx2, and that the activity of...

Tissue Engineering Part A, Sep 1, 2018

Complications that arise from impaired fracture healing have considerable socioeconomic implicati... more Complications that arise from impaired fracture healing have considerable socioeconomic implications. Current research in the field of bone tissue engineering predominantly aims to mimic the mature bone tissue microenvironment. This approach, however, may produce implants that are intrinsically unresponsive to the cues present during the initiation of fracture repair. As such, this study describes the development of decellularised xenogeneic hyaline cartilage matrix in an attempt to mimic the initial reparative phase of fracture repair. Three approaches based on vacuum-assisted osmotic shock (Vac-OS), Triton X (Vac-Stx) and SDS (Vac-SDS) were investigated. The Vac-OS methodology reduced DNA content below 50ng/mg of tissue, whilst retaining 85% of the sGAG content and as such was selected as the optimal methodology for decellularisation. The resultant Vac-OS scaffolds (dcECM) were also devoid of the immunogenic alpha-gal epitope. Furthermore, minimal disruption to the structural integrity of the dcECM was demonstrated using differential scanning calorimetry (DSC) and fluorescence lifetime imaging microscopy (FLIM). The biological integrity of the dcECM was confirmed by its ability to drive the chondrogenic commitment and differentiation of human chondrocytes and periosteumderived cells respectively. Furthermore, histological examination of dcECM constructs implanted in immunocompetent mice revealed a predominantly M2-macrophage driven regenerative response both at 2 and 8 weeks post-implantation. These findings contrasted with the implanted native costal cartilage that elicited a predominantly M1-macrophage mediated inflammatory response. This study highlights the capacity of dcECM from the Vac-OS methodology to direct the key biological processes of endochondral ossification, thus potentially recapitulating the callus phase of fracture repair.

JBMR Plus

The identity of the cells that form the periosteum during development is controversial with curre... more The identity of the cells that form the periosteum during development is controversial with current dogma suggesting these are derived from a Sox9-positive progenitor. Herein, we characterize a newly created Prrx1eGFP reporter transgenic mouse line during limb formation and postnatally. Interestingly, in the embryo Prrx1eGFP-labeled cells become restricted around the Sox9-positive cartilage anlage without themselves becoming Sox9-positive. In the adult, the Prrx1eGFP transgene live labels a subpopulation of cells within the periosteum that are enriched at specific sites, and this population is diminished in aged mice. The green fluorescent protein (GFP)-labeled subpopulation can be isolated using fluorescence-activated cell sorting (FACS) and represents approximately 8% of all isolated periosteal cells. The GFP-labeled subpopulation is significantly more osteogenic than unlabeled, GFP-negative periosteal cells. In addition, the osteogenic and chondrogenic capacity of periosteal cells in vitro can be extended with the addition of fibroblast growth factor (FGF) to the expansion media. We provide evidence to suggest that osteoblasts contributing to cortical bone formation in the embryo originate from Prrx1eGFP-positive cells within the perichondrium, which possibly piggyback on invading vascular cells and secrete new bone matrix. In summary, the Prrx1eGFP mouse is a powerful tool to visualize and isolate periosteal cells and to quantify their properties in the embryo and adult.

Journal of Clinical Oncology

e19505 Background: Receptor tyrosine kinase-like Orphan Receptor-1 (ROR1) is widely expressed on ... more e19505 Background: Receptor tyrosine kinase-like Orphan Receptor-1 (ROR1) is widely expressed on hematological and solid tumors. NVG-111, a first in class humanized tandem scFv ROR1xCD3 bispecific antibody elicits potent killing of ROR1+ tumor cells in vitro and in vivo. This bispecific T-cell engager (TCE) is being evaluated in a first in human, Phase I trial in patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The predicted therapeutic dose and steady state serum concentrations of NVG-111 were estimated by allometric scaling using relevant doses from a murine PK study. To assess free drug levels in patients following 21 days of continuous infusion of NVG-111, a bespoke, sensitive pharmacokinetics (PK) assay with high levels of specificity and sensitivity was developed. Methods: Anti-idiotype (anti-ID) antibodies directed to anti-ROR1 (αROR1-ID) and anti-CD3 (αCD3-ID) were generated by mouse immunization or by phage display from cu...

International Journal of Experimental Pathology, 2015

Journal of Endocrinology, 2012

AMP-activated protein kinase (AMPK) is a key regulator of cellular and body energy homeostasis. W... more AMP-activated protein kinase (AMPK) is a key regulator of cellular and body energy homeostasis. We previously demonstrated that AMPK activation in osteoblasts increases in vitro bone formation while deletion of the Ampkα1 (Prkaa1) subunit, the dominant catalytic subunit expressed in bone, leads to decreased bone mass in vivo. To investigate the cause of low bone mass in the Ampkα1−/− mice, we analysed bone formation and resorption in the tibia of these mice by dynamic histomorphometry and determined whether bone turnover can be stimulated in the absence of the Ampkα1 subunit. We subjected 12-week-old Ampkα1+/+ and Ampkα1−/− mice to ovariectomy (OVX), intermittent PTH (iPTH) administration (80 μg/kg per day, 5 days/week) or both OVX and iPTH hormonal challenges. Tibiae were harvested from these mice and bone micro-architecture was determined by micro-computed tomography. We show for the first time that Ampkα1−/− mice have a high bone turnover at the basal level in favour of bone reso...

Acta Biomaterialia, 2019

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service... more This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Osteomimetic matrix components alter cell migration and drug response in a 3D tumourengineered osteosarcoma model.

Bioengineering

The control of cell behaviour in an effort to create highly homogeneous cultures is becoming an a... more The control of cell behaviour in an effort to create highly homogeneous cultures is becoming an area of intense research, both to elucidate fundamental biology and for regenerative applications. The extracellular matrix (ECM) controls many cellular processes in vivo, and as such is a rich source of cues that may be translated in vitro. Herein, we describe the creation of cell culture coatings from porcine decellularised hyaline cartilage through enzymatic digestion. Surprisingly, heat-mediated sterilisation created a coating with the capacity to rapidly and robustly induce chondrogenic differentiation of human periosteal cells. This differentiation was validated through the alteration of cell phenotype from a fibroblastic to a cuboidal/cobblestone chondrocyte-like appearance. Moreover, chondrogenic gene expression further supported this observation, where cells cultured on heat sterilised ECM-coated plastic displayed higher expression of COL2A1, ACAN and PRG4 (p < 0.05) compared ...

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

smokeinduced induction of antioxidant enzyme activities in airway leukocytes is absent in active ... more smokeinduced induction of antioxidant enzyme activities in airway leukocytes is absent in active smokers with COPD

Journal of Clinical Oncology

7535 Background: NVG-111 is a first in class, humanized, tandem scFv ROR1xCD3 bispecific T cell e... more 7535 Background: NVG-111 is a first in class, humanized, tandem scFv ROR1xCD3 bispecific T cell engager that mediates potent killing of ROR1+ tumours by engaging an epitope on the Frizzled domain of ROR1 and redirecting T cell activity via the CD3 binder. Methods: This phase I/II study is evaluating NVG-111 in patients with relapsed/refractory (R/R) CLL and MCL who have received ≥2 prior systemic therapies and have achieved a stable, or partial response to the last line of therapy. NVG-111 was delivered as continuous intravenous (cIV) infusion over 21 days per cycle, with each patient typically receiving 3 cycles of treatment. The first 3 single patient cohorts were subjected to accelerated dose titration (ADT) over a range of 0.3-30µg/day. Dose-escalation steps in the subsequent, multi-patient, cohorts were determined using a continuous reassessment method with overdose control. The primary endpoints are safety and determination of MTD/RP2D. Secondary objectives are pharmacokinetic...

Journal of Orthopaedic Translation, 2021

Background: Sclerosteosis, a severe autosomal recessive sclerosing skeletal dysplasia characteris... more Background: Sclerosteosis, a severe autosomal recessive sclerosing skeletal dysplasia characterised by excessive bone formation, is caused by absence of sclerostin, a negative regulator of bone formation that binds LRP5/6 Wnt co-receptors. Current treatment is limited to surgical management of symptoms arising from bone overgrowth. This study investigated the effectiveness of sclerostin replacement therapy in a mouse model of sclerosteosis. Methods: Recombinant wild type mouse sclerostin (mScl) and novel mScl fusion proteins containing a C-terminal human Fc (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), were produced and purified using mammalian expression and standard chromatography methods. In vitro functionality and efficacy of the recombinant proteins were evaluated using three independent biophysical techniques and an in vitro bone nodule formation assay. Pharmacokinetic properties of the proteins were investigated in vivo following a single administration to young female wild type (WT) or SOST knock out (SOST-/-) mice. In a six week proof-of-concept in vivo study, young female WT or SOST-/mice were treated with 10 mg/kg mScl hFc or mScl hFc PD (weekly), or 4.4 mg/kg mScl (daily). The effect of recombinant sclerostin on femoral cortical and trabecular bone parameters were assessed by micro computed tomography (μCT). Results: Recombinant mScl proteins bound to the extracellular domain of the Wnt co-receptor LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in vitro. Pharmacokinetic assessment following a single dose administered to WT or SOST-/mice indicated the presence of hFc increased protein half-life from less than 5 min to at least 1.5 days. Treatment with mScl hFc PD over a six week period resulted in modest but significant reductions in trabecular volumetric bone mineral density (vBMD) and bone volume fraction (BV/TV), of 20% and 15%, respectively. Conclusion: Administration of recombinant mScl hFc PD partially corrected the high bone mass phenotype in SOST-/mice, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/mouse model of sclerosteosis. The translational potential of this article: These findings support the concept that exogenous sclerostin can reduce bone mass, however the modest efficacy suggests that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored.

RMD Open, 2020

ObjectivesInterleukin (IL)-17 signalling has been shown to be a key regulator of disease in ankyl... more ObjectivesInterleukin (IL)-17 signalling has been shown to be a key regulator of disease in ankylosing spondylitis (AS) with several IL-17 blockers currently clinically approved. Despite this, the role of IL-17 in bone pathology is poorly understood. This study aimed to investigate IL-17 signalling in the context of pathological bone formation.MethodsA biomimetic human periosteum-derived cell (hPDC) model of osteogenic differentiation was used in combination with recombinant IL-17 cytokines, T-cell supernatants or serum from patients with AS. IL-17A, IL-17F and bimekizumab monoclonal antibodies were used to block IL-17 cytokine action.ResultsRecombinant IL-17A and IL-17F are pro-osteogenic with respect to hPDC differentiation. T helper 17 or γδ-T cell supernatants also potently stimulated in vitro bone formation, which was blocked deeper by dual inhibition of IL-17A and IL-17F than by neutralisation of IL-17A or IL-17F individually. Osteogenic blockade may be due to an increase in e...

Cytotherapy, 2020

Background: The periosteum is a highly vascularized, collagen-rich tissue that plays a crucial ro... more Background: The periosteum is a highly vascularized, collagen-rich tissue that plays a crucial role in directing bone repair. This is orchestrated primarily by its resident progenitor cell population. Indeed, preservation of periosteum integrity is critical for bone healing. Cells extracted from the periosteum retain their osteochondrogenic properties and as such are a promising basis for tissue engineering strategies for the repair of bone defects. However, the culture expansion conditions and the way in which the cells are reintroduced to the defect site are critical aspects of successful translation. Indeed, expansion in human serum and implantation on biomimetic materials has previously been shown to improve in vivo bone formation. Aim: This study aimed to develop a protocol to allow for the expansion of human periosteum derived cells (hPDCs) in a biomimetic periosteal-like environment. Methods: The expansion conditions were defined through the investigation of the bioactive cues involved in augmenting hPDC proliferative and multipotency characteristics, based on transcriptomic analysis of cells cultured in human serum. Results: Master regulators of transcriptional networks were identified, and an optimized periosteum-derived growth factor cocktail (PD-GFC; containing b-estradiol, FGF2, TNFa, TGFb, IGF-1 and PDGF-BB) was generated. Expansion of hPDCs in PD-GFC resulted in serum mimicry with regard to the cell morphology, proliferative capacity and chondrogenic differentiation. When incorporated into a three-dimensional collagen type 1 matrix and cultured in PD-GFC, the hPDCs migrated to the surface that represented the matrix topography of the periosteum cambium layer. Furthermore, gene expression analysis revealed a down-regulated WNT and TGFb signature and an up-regulation of CREB, which may indicate the hPDCs are recreating their progenitor cell signature. Conclusion: This study highlights the first stage in the development of a biomimetic periosteum, which may have applications in bone repair.

Development, 2019

In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which ... more In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which is characterised, in part, by craniofacial abnormalities. Here, we analyse the role of Dchs1-Fat4 signalling during osteoblast differentiation in mouse. We show that Fat4 and Dchs1 mutants mimic the craniofacial phenotype of the human syndrome and that Dchs1-Fat4 signalling is essential for osteoblast differentiation. In Dchs1/Fat4 mutants, proliferation of osteoprogenitors is increased and osteoblast differentiation is delayed. We show that loss of Dchs1-Fat4 signalling is linked to increased Yap-Tead activity and that Yap is expressed and required for proliferation in osteoprogenitors. In contrast, Taz is expressed in more-committed Runx2-expressing osteoblasts, Taz does not regulate osteoblast proliferation and Taz-Tead activity is unaffected in Dchs1/Fat4 mutants. Finally, we show that Yap and Taz differentially regulate the transcriptional activity of Runx2, and that the activity of...