Repopulation of vascularized bone allotransplants with recipient-derived cells

Detection by laser capture microdissection and real-time PCR

Michael Pelzer, Mikko Larsen, Patricia F. Friedrich, Ross A. Aleff, Allen Thorp Bishop

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Mechanisms underlying successful composite tissue transplantation must include an analysis of transplant chimerism, which is little studied, particularly in calcified tissue. We have developed a new method enabling determination of lineage of selected cells in our model of vascularized bone allotransplantation. Vascularized femoral allotransplantation was performed from female Dark Agouti (DA) donor rats to male Piebald Virol Glaxo (PVG) recipients, representing a major histocompatibility mismatch. Four groups differed in use of immunosuppression (±2 weeks Tacrolimus) and surgical revascularization, by implantation of either a patent or a ligated saphenous arteriovenous (AV) bundle. Results were assessed at 18 weeks. Bone blood flow was measured by the hydrogen washout technique and transverse specimens were prepared for histology. Real-time PCR was performed on DNA from laser capture microdissected cortical bone regions to determine the extent of chimerism. To do so, we analyzed the relative expression ratio of the sex-determining region Y (Sry) gene, specific only for recipient male rat DNA, to the cyclophilin housekeeper gene. Substantial transplant chimerism was seen in cortical bone of all groups (range 77-97%). Rats without immunosuppression and with a patent AV bundle revealed significantly higher chimerism than those with immunosuppression and a ligated AV bundle, which maintained transplant cell viability. We describe a new method to study the extent of chimerism in rat vascularized bone allotransplants, including a sex-mismatched transplantation model, laser capture microdissection of selected bone regions, and calculation of the relative expression ratio.

Original languageEnglish (US)
Pages (from-to)1514-1520
Number of pages7
JournalJournal of Orthopaedic Research
Volume27
Issue number11
DOIs
StatePublished - Nov 2009

Fingerprint

Laser Capture Microdissection
Chimerism
Real-Time Polymerase Chain Reaction
Bone and Bones
Immunosuppression
Transplants
sry Genes
Cyclophilins
Tissue Transplantation
Histocompatibility
DNA
Tacrolimus
Thigh
Hydrogen
Cell Survival
Histology
Lasers
Transplantation
Genes

Keywords

  • Allotransplant
  • Bone
  • Chimerism
  • Microdissection
  • Real-time PCR

ASJC Scopus subject areas

  • Orthopedics and Sports Medicine

Cite this

Repopulation of vascularized bone allotransplants with recipient-derived cells : Detection by laser capture microdissection and real-time PCR. / Pelzer, Michael; Larsen, Mikko; Friedrich, Patricia F.; Aleff, Ross A.; Bishop, Allen Thorp.

In: Journal of Orthopaedic Research, Vol. 27, No. 11, 11.2009, p. 1514-1520.

Research output: Contribution to journalArticle

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