B ONE VASCULARIZATION

The vascularized component of engineered bone graft is critical for its survival and function after in vivo implantation. This major concern has led to the development of several studies in order to 1) achieve a vascularized bone graft and 2) understand the cross-talk between bone and vascular cells. Table II summarizes most recent findings in this domain.

Table II – Co-culture and single-cell approaches for bone vascularization. Compiled from references

[36-42]

Co-culture approach Scaffold In vitro evaluation In vivo evaluation Ref. HDMEC

1.3x105

HOb 0.2x105

Silk fibroin

nets 7 and 14 days [43] SCID mice, subcutaneous 14 days

in vitro pre-formed microcapillary anastomosed with host vasculature

[36]

HUVEC

(1:2) HOP (1:2) Alginate micro- spheres

Dynamic culturing, 3 weeks Upregulation of AP and OCN

GE and increased

mineralization deposits; Increased VEGF secretion

SCID mice, femur defect 3 and 6 weeks

Mineralization significantly increased when HOPs were co-immobilized with HUVECs

[37]

HUVEC

(1:1) hMSC (1:1) n/a DMEM + M199 (1:1 v/v), 3 weeks Promotion of osteogenic differentiation and significant increase on hMSC proliferation n/a [38] HUVEC 1x106 hMSC 2.5x105 Collagen-

(4:1) (4:1) gel + PLGA scaffold

Scaffolds were then seeded with collagen-fibronectin gel containing HUVEC and MSC and implanted subcutaneously in SCID mice

HUVECs formed tube-like structures and networks 4–7 days after implantation; anastomosis occurred by day 11;

At 4 weeks, network was mature and mineralization occured

cEPC cMSC Collagen fiber mesh scaffold

n/a SCID mice, subcutaneous

12 weeks

Co-implanted EPCs increased neovascularization;

Bone area was greater in the MSC+EPC group [40] HUVEC 1x105 ₊ 2x105 hMSC 1x106 Fibrin/ Matrigel + Tutobone hMSC osteoinduction,

3 days SCID mice, orthotopic calvarial defect, 6 weeks HUVECs formed complex 3D

networks of perfused neovessels; MSCs supported bone formation [41] Single-cell approach hASC 1x106 cells/cm 2 - Hydroxy- apatite scaffold Co-differentiation, 3 weeks M199 + osteogenic and endothelial GF

Nude rats, calvarial defect, 3 weeks

ASC adhesion, distribution,

proliferation and GE

demonstrated a full osteogenic and vasculogenic commitment

in vitro and in vivo

[42]

AP – alkaline phosphatase; cEPC – canine endothelial progenitor cells; cMSC - canine mesenchymal stem cells; DMEM - Dulbecco's Modified Eagle's medium; GE – gene expression; GF – growth factors; hASC – human adipose stem cells; HDMEC - human dermal microcapillary endothelial cells; hMSC - human mesenchymal stem cells; HOb – human osteoblasts; HOP- human osteoprogenitors; HUVEC - human umbilical vein endothelial cells; M199 – medium; n/a – non applicable; OCN – osteocalcin; PLGA - poly(DL -lactide-co-glycolide); SCID – severe combined immunodeficiency; VEGF - vascular endothelial growth factor; v/v - volume/volume

A clear trend is observed by analyzing Table II: in the last three years, HUVECs and hMSC interactions have been the most thoroughly explored in order to respectively develop vascular and osteogenic compartments. This is an evolution relatively to earlier studies, well reviewed by Santos and Reis 2010 [44] when HDMECs and human osteoblasts (hOb) were the most used cell sources.

HUVECs are a key cell source on what concerns the formation of functional vascular structures. While HDMEC develop microvascular structures, HUVECs are capable of forming

Chapter II – Engineering bone: current considerations

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large blood vessels, which is a beneficial characteristic on aiming to engineer stable functional vascularized bone grafts.

hMSC are a more advantageous cell type than hOb, once MSCs have the potential to undergo two main roles: 1) as osteogenic progenitors, forming mineralized bone matrix [34, 35]; and 2) as pericytes, responsible for stabilizing vascular networks. In this regard, recent publications have been supporting this fact, majorly when MSC are co-cultured with mature endothelial cells [39, 41]. Data summarized in Table II may support this vision: in most studies, better outcomes for both tissue compartments were obtained when endothelial cells were co-immobilized/co- implanted with osteo-progenitors [37, 40].

The close interaction between MSCs and HUVECs in co-culture systems is yet target of extensive research [38, 39, 41]. A reliable in vitro model composed of these cells, could become a great tool to clarify and specify the molecular mechanisms that regulate inter-cellular interactions, relevant not only in the scope of engineering vascularized bone grafts, but also to provide a platform to study disease conditions and/or developmental processes.

On the other hand, given the final therapeutic aim, hASC present a clear lead over the HUVEC- hMSC approach, once the same cell source is used to fully develop the vascularized bone graft. hASC have proven to be no less capable of osteogenic differentiation and bone tissue development than bone-marrow MSC [32, 45], which confirms as a promising alternative for cell based therapies of bone [46]. In addition, hASC have demonstrated its intrinsic potential for endothelial differentiation and capillary formation [47-49]. As mentioned in table II, when nourished with a cocktail of both osteogenic and vasculogenic growth factors, hASC show to commit to both lineages, by expressing correspondent specific markers, such as osteopontin, osteonectin, osteocalcin, and collagen type I, for bone; and CD31, von Willebrand Factor and VEGF, for vasculogenesis [42]. Besides this potential, hASC present other key characteristics such as:

a. Adipose tissue (AT) is harvested from the patient by either dedicated or non-dedicated procedures. AT can be obtained repeatedly in large quantities under local anesthesia and with a minimum of patient discomfort;

b. Can be used used in an autologous manner, but still presents a high potential for allogeinic purposes;

c. Adipose tissue yields very high cell numbers, as compared to bone-marrow harvest procedures [50].

Although hASC present such attractive characteristics, the major challenge - to fully engineer functional bone and vessels concurrently - is yet to be achieved.