The process entails the condensation (clustering together through cell surface receptors and adhesion molecules ) of chondrocytes, which secrete a collagenous (type II) matrix rich in proteoglycans. but indistinguishable from surrounding bone. However, in certain circumstances, the defect is too large (due to tumour resection, osteomyelitis, atrophic nonunions, and periprosthetic bone loss), or the underlying physiological state of the patient impairs natural healing (osteoporosis, infection, diabetes, and smoking) necessitating intervention. Autologous bone grafting is today the gold standard for bone repair, although the costs of this approach are considerable due to the additional surgical procedures required to harvest the bone material, the consequent donor site morbidity , and the risk of infection and complications. Additionally, this approach is hampered by Vecabrutinib the limited amount of donor material available for transplantation which can be prohibitive when dealing with large defects. To resolve these issues, both allograft- and xenograft-based strategies have been proposed; however the risk of rejection in the Vecabrutinib former and of zoonoses in the latter has reduced their clinical impact. Bone tissue engineering (BTE) is an alternative strategy that has been explored to fill the clinical need for autologous bone transplantation. Almost half a century has passed since the demonstration that ectopic transplantation of bone marrow and bone fragments leads to the formation ofde novo in vitroand can regenerate fully functional bone organsin vivois well accepted, although the identity and precise molecular characterisation of the cell population responsible FLJ31945 are still a matter of study and debate (reviewed in [4, 5]). Theex vivoexpansion and manipulation of stromal cells derived from various sources form the foundation of the majority of current bone tissue engineering attempts to meet the clinical demands for bone regeneration and repair. Over the last 50 years, the BTE field has made significant advances towards overcoming the limitations of conventional methods which is particularly relevant when an underlying pathology calls for alternatives to thestatus quo(TGF-in vitrooptimisation of treatments as a means to supportin vivo in vivoregeneration and spatial organisation of skeletal tissues. In the early 1990s Arnold Caplan’s group showed that rat bone marrow-derived mesenchymal cells, purified through plastic adherence, could be passaged multiple times, demonstrating self-renewal (albeitin vitroin vivoin vitroself-renewal, giving rise to secondary colonies upon replating at the clonal level [41, 42]. demonstration of BMSC stem cell characteristics, namely, self-replication and multipotency, came with the description of CD146+/MCAM (melanoma cell adhesion molecule)  and nestin+  perivascular adventitial cells. Transplantation of single CFU-f-derived CD146+ colonies implanted in hydroxyapatite-tricalcium phosphate (HA-TCP) carrier in a fibrin gel in mice resulted in the formation of ossicles with a functional bone marrow populated by murine (host) haematopoietic cells and endothelium with human CD146+ adventitial cells lining the sinusoidal vessels, which were capable of generating secondary CFU-fsin vitro. Similarly, implantation of nestin+ clonal cell spheres harvested two months after subcutaneous implantation in mice resulted in the generation of secondary ossicles with donor-derived osteoblasts and nestin+ cells after eight months . Nestin+ cells were shown to spatially associate with haematopoietic stem cells (HSCs), to express high levels Vecabrutinib of HSC maintenance genes, and to influence HSC homing in addition to differentiation into osteochondral lineages; in addition they were shown to be entirely responsible for the clonogenic activity of the CD45? cell fraction . More recently, evidence for a skeletal stem cell (SSC) resident in the BM reticulum, characterised by expression of the BMP antagonist Gremlin-1, has emerged  which has challenged previous ideas about the identity of the SSC, particularly the use of nestin as an appropriate SSC marker and the developmental origins of BM adipocytes , although it is possible that these conflicting data may be due to different active populations of SSCs during different phases of development [45, 46]. 2.2. Clinical BTE Application of BMSCs Practically, BMSCs are applicable to large bone defects in both small  and large [48, 49] animals when implanted within hydroxyapatite-based Vecabrutinib scaffolds. Experimental evidence for the ability of BMSCs to repair bone defects was given crucial clinical support in 2001, when Quarto and colleagues published results obtained in three patients with various long bone defects . BMSCs were isolated and expandedex vivounder the stimulation Vecabrutinib of specific growth factors  before implantation on hydroxyapatite (HA) scaffolds tailored.