Potential role of pre-existing blood vessels for vascularization and mineralization of osteochondral graftsAn intravital microscopic study in mice

Abstract

Background The aim of this study was to develop an experimental model that allows to elude the potential role of the preexisting graft microvasculature for vascularization and mineralization of osteochondral grafts.Animals and methods For that purpose, the II-IV metatarsals of fetal DDY-mice known to be nonvascularized at day 16 of gestation (M16) but vascularized at day 18 (M18) were freshly transplanted into dorsal skin fold chambers of adult DDY mice. Using intravital microscopy angiogenesis, leukocyte-endothelium interaction and mineralization were assessed for 12 days.Results Angiogenesis occurred at 32 hours in M18, but not before 57 hours in M16 (p = 0.002), with perfusion of these vessels at 42 hours (p = 0.005) and 65 hours (p = 0.1), respectively. Vessels reached a density three times as high as that of the recipient site at day 6, remaining constant until day 12 in M18, whereas in M16 vascular density increased from day 6 and reached that of M18 at day 12 (p = 0.04). Leukocyte-endothelium interaction showed sticker counts elevated by a factor of 4-5 in M18 as compared to M16. Mineralization of osteochondral grafts did not differ between M16 and M18, which significantly increased in both groups throughout the observation period.Interpretation We propose the faster kinetics in the angiogenic response to M18 and the elevated counts of sticking leukocytes to rest on the potential of establishing end-to-end anastomoses (inosculation) of the vascularized graft with recipient vessels.

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“…Following implantation, angiogenesis, vessel perfusion and increased vascular density occurred earlier in M18 compared to M16 metatarsals, due to the preexisting vessels in the fresh osteochondral grafts contributing to revascularisation by inosculation. In contrast, M16 grafts established a microvasculature exclusively by active de novo synthesis . Therefore, vascularisation data from our model are in agreement with previously published studies.…”

“…There is formation of blind ended vessels after 24 hr, with functional vessels exhibiting blood flow by 48 hr. This is in agreement with previous studies demonstrating blood vessel development commencing by 48 hr, with vascular perfusion by 96 hr, in engrafted femurs, implanted immediately following resection . However, initiation of the microvascular architecture is slower in bones undergoing cryopreservation or boiling, indicating the importance of using freshly harvested bone for such studies.…”

“…Three middle metatarsals (II–IV) from each new born mouse paw were removed for implantation into the DSC following cull . In brief, paws were placed in 70% ethanol for 5 min, and then blunt dissected to remove the soft tissues in DMEM media at room temperature.…”

“…Vascular density of perfused and nonperfused vessels was measured over the surface area of the graft, presented as mm/mm 2 and quantified using the public domain NIH image analysis programme http://rsb.info.nih.gov/nih-image/ . Perfusion was measured by administering FITC‐BSA (0.25 ml/kg) via i.v.…”

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“…Osteochondral autograft transfers is another technique developed to fill osteoarticular defects in weight bearing regions of the knee [15-16]. However, chondrocyte death at the margins of the autograft [17-20] and persistent gaps between the graft and the host tissue [21-24] have been shown to lead to poor graft durability over time [25]. In cases where implants are used to fill the defect, margin integration is frequently characterized by gaps and fissuring in histologic sections [26-32].…”