Characterization of Cells Isolated from Genetic and Trauma-Induced Heterotopic Ossification

<div><p>Heterotopic ossification (HO) is the pathologic formation of bone separate from the normal skeleton. Although several models exist for studying HO, an understanding of the common <i>in vitro</i> properties of cells isolated from these models is lacking. We studied three separate animal models of HO including two models of trauma-induced HO and one model of genetic HO, and human HO specimens, to characterize the properties of cells derived from tissue containing pre-and mature ectopic bone in relation to analogous mesenchymal cell populations or osteoblasts obtained from normal muscle tissue. We found that when cultured <i>in vitro</i>, cells isolated from the trauma sites in two distinct models exhibited increased osteogenic differentiation when compared to cells isolated from uninjured controls. Furthermore, osteoblasts isolated from heterotopic bone in a genetic model of HO also exhibited increased osteogenic differentiation when compared with normal osteoblasts. Finally, osteoblasts derived from mature heterotopic bone obtained from human patients exhibited increased osteogenic differentiation when compared with normal bone from the same patients. These findings demonstrate that across models, cells derived from tissues forming heterotopic ossification exhibit increased osteogenic differentiation when compared with either normal tissues or osteoblasts. These cell types can be used in the future for <i>in vitro</i> investigations for drug screening purposes.</p></div

Characterization of <i>in vitro</i> osteogenic differentiation of osteoblasts isolated from a genetic model of HO in mice.

<p>(A) Osteoblasts were harvested and cultured from ectopic bone produced in mice with a constitutively active form of the BMP receptor ALK2 in cells expressing NFAT and from normal bone of WT mice. Cells harvested from the mutant mice (NF-HO) showed more ALP activity and more in-vitro bone deposition by Alizarin red staining than cells derived from WT mice (WT Ob). Representative wells and 10x micrographs are shown. (B) Similarly, these cells from the mice with constitutively active ACVR1 demonstrated increased expression of osteogenic gene transcripts for <i>Ocn</i>, <i>Opn</i>, <i>Osx</i>, and <i>Runx2</i>. (C) Western blot analysis of pSmad 5 protein levels in these cells after 0, 6, and 18 days in osteogenic media. Data shown are reported as the mean ± SD of at least three separate experimental samples performed in triplicate. *<i>p</i><0.05; **<i>p</i><0.01. Student T-test.</p

FACS analysis of early post-injury tissue and HO osteoblasts.

<p>(A) FACS gating for osteoproliferative cells from tendon transection site one week after injury and contralateral hindlimb; (B) Bar graph showing quantification; (C) FACS gating for osteoproliferative cells from HO and normal osteoblasts of mutant mice and wild type osteoblasts; (D) Bar graph showing quantification. All experiments performed in triplicate. * = p<0.05.</p

Characterization of <i>in vitro</i> osteogenic differentiation of muscle-derived MSCs isolated from a mouse tenotomy/burn model of HO.

<p>(A) MSCs were harvested and cultured from the soft tissue about the ankle in a mouse tenotomy/burn model that demonstrates ectopic bone development 4–6 weeks after injury. MSCs harvested from the tenotomy site (Tenotomy) 5 days after injury showed more ALP activity and more in-vitro bone deposition by Alizarin red staining than cells derived from the same site on the contralateral, un-injured leg (Control). Representative wells and 10x micrographs are shown. (B) Gene transcript levels of <i>Ocn</i>, <i>Opn</i>, <i>Osx</i>, and <i>Runx2</i>. (C) Western blot analysis of pSmad 1/5 protein levels in these cells after 0, 6, and 18 days in osteogenic media. Data shown are reported as the mean ± SD of at least three separate experimental samples performed in triplicate. *<i>p</i><0.05; **<i>p</i><0.01. Student T-test.</p

Characterization of <i>in vitro</i> osteogenic differentiation of osteoblasts isolated from HO and normal bone.

<p>(A) Human osteoblasts were harvested from excised HO (HO-Ob) and surrounding normal bone (Ob). HO derived osteoblasts showed more ALP activity and more in-vitro bone deposition by Alizarin red staining than cells derived from normal bone. Representative wells and 10x micrographs are shown. (B) Gene transcript levels of <i>Ocn</i>, <i>Osx</i>, and <i>Runx2</i>. (C) Western blot analysis of pSmad 5 protein levels in these cells after 0, 6, and 18 days in osteogenic media. Data shown are reported as the mean ± SD of at least three separate experimental samples performed in triplicate. *<i>p</i><0.05; **<i>p</i><0.01. Student T-test.</p

Characterization of osteoblasts and MSC <i>in vitro</i> cell proliferation using cells isolated and expanded from 4 sources of HO tissue.

<p>(A) Human osteoblasts cultured from heterotopic bone (HO-Ob) demonstrated higher proliferation rates by cell counting (left) and BrDU (right) than osteoblasts derived from normal bone (Ob). (B) Osteoblasts from a genetic model of HO (NF-HO) in which the BMP receptor demonstrated higher proliferation rates compared to osteoblasts from WT mice (WT Ob) by cell counting (left) and BrDU (right). (C) In a mouse tenotomy/burn model of HO, mesenchymal cells derived from soft tissue harvested from the tenotomy site (Tenotomy) demonstrated higher proliferation than cells taken from soft tissue from the contralateral, non-tenotomized leg (Control) by cell counting (left) and BrDU (right). (D) In a blast/polytraumatic extremity injury model in rats, osteoblasts from the injured side (Injured) were more proliferative than cells from the non-injured side (control). Cell proliferation was determined by manual hemocytometer cell counts using Trypan blue stain (left) and BrDU incorporation (right). Data are means ± SD. *<i>p</i><0.05; **<i>p</i><0.01. Student T-test.</p

Characterization of <i>in vitro</i> osteogenic differentiation of muscle-derived MSC isolated from a rat blast/polytraumatic extremity injury model of HO.

<p>(A) MSCs were harvested and cultured from the soft tissue about the injury site in a rat blast/ polytraumatic extremity injury model. MSCs harvested from the blast injury/amputation site (Injured) 5 days after injury showed more ALP activity and more in vitro bone deposition by Alizarin red staining than cells derived from the same site on the contralateral, un-injured limb (Control). Representative wells and 10x micrographs are shown. (B) Expression levels of <i>Opn</i>, <i>Osx</i>, and <i>Runx2</i>. (C) Western blot analysis of pSmad 1/5 protein levels in these cells after 0, 6, and 18 days in osteogenic media. Data shown are reported as the mean ± SD of at least three separate experimental samples performed in triplicate. *<i>p</i><0.05; **<i>p</i><0.01. Student T-test.</p