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Circulating osteogentic precursor cells in non-hereditary heterotopic ossification
BON-11517; No. of pages: 5; 4C: Bone xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bone journal homepage: www.elsevier.com/locate/bone
Review Article
Circulating osteogentic precursor cells in non-hereditary heterotopic ossification Kevin P. Egan a, Gustavo Duque b,c, Mary Ann Keenan d, Robert J. Pignolo e,⁎ a
Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States Australian Institute for Musculoskeletal Science (AIMSS), The University of Melbourne and Western Health, Melbourne, VIC, Australia Department of Medicine-Western Health, Melbourne Medical School, The University of Melbourne, Melbourne, VIC, Australia d Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States e Department of Medicine, Mayo Clinic School of Medicine, Mayo Clinic, Rochester, MN, United States b c
a r t i c l e
i n f o
Article history: Received 21 December 2017 Accepted 29 December 2017 Available online xxxx Keywords: Heterotopic ossification Injury Traumatic brain injury Cerebrovascular accident Trauma Spinal cord injury Circulating osteogenic precursor (COP) cells
a b s t r a c t Non-hereditary heterotopic ossification (NHHO) may occur after musculoskeletal trauma, central nervous system (CNS) injury, or surgery. We previously described circulating osteogenic precursor (COP) cells as a bone marrow–derived type 1 collagen+ CD45+ subpopulation of mononuclear adherent cells that are able of producing extraskeletal ossification in a murine in vivo implantation assay. In the current study, we performed a tissue analysis of COP cells in NHHO secondary to defined conditions, including traumatic brain injury, spinal cord injury, cerebrovascular accident, trauma without neurologic injury, and joint arthroplasty. All bone specimens revealed the presence of COP cells at 2–14 cells per high power field. COP cells were localized to early fibroproliferative and neovascular lesions of NHHO with evidence for their circulatory status supported by their presence near blood vessels in examined lesions. This study provides the first systematic evaluation of COP cells as a contributory histopathological finding associated with multiple forms of NHHO. These data support that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites, such as those subject to soft tissue injury, and due to their migratory nature, may likely be involved in seeding sites distant to CNS injury. © 2018 Elsevier Inc. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . 2.1. Patients . . . . . . . . . . . . . . . . . 2.2. Specimen retrieval . . . . . . . . . . . . 2.3. Tissue preparation and basic staining . . . 2.4. Immunofluorescence studies . . . . . . . 2.5. Quantitation of COP cells in tissue specimens 3. Results . . . . . . . . . . . . . . . . . . . . 3.1. COP cells are present in lesions of NHHO . . 3.2. COP cells are found in early stage lesions . . 4. Discussion . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . Funding. . . . . . . . . . . . . . . . . . . . . . . Authors' roles . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
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⁎ Corresponding author at: Division of Geriatric Medicine & Gerontology, Robert and Arlene Kogod Professor of Geriatric Medicine, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, United States. E-mail addresses: [email protected] (K.P. Egan), [email protected] (G. Duque), [email protected] (R.J. Pignolo).
https://doi.org/10.1016/j.bone.2017.12.028 8756-3282/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: K.P. Egan, et al., Circulating osteogentic precursor cells in non-hereditary heterotopic ossification, Bone (2018), https:// doi.org/10.1016/j.bone.2017.12.028
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K.P. Egan et al. / Bone xxx (2018) xxx–xxx
Table 1 COP cells in early stage NHHO lesions. Diagnosis
Age
Sex
COP cells per HPF (%)
Stage of lesion
TBI
36 40 53 25 42 21 30 38 53 52 80 84 44
M M M M M M M M M M F F F
2 7 6 4 8 6 3 14 2 5 6 2 7
Fibroproliferative Fibroproliferative Fibroproliferative Fibroproliferative Neovascular Fibroproliferative Fibroproliferative Fibroproliferative Fibroproliferative Neovascular Fibroproliferative Fibroproliferative Neovascular
SCI Trauma
Arthroplasty
CVA
HPF, high power field; TBI, traumatic brain injury; SCI, spinal cord injury; CVA, cerebrovascular accident.
Newly formed bone can lead to significant loss of motion at susceptible joints or contribute to stenosis in affected heart valves [1,2]. We previously described circulating osteogenic precursor (COP) cells as a bone marrow–derived type 1 collagen (Col1)+ CD45+ subpopulation of mononuclear adherent cells that are able to produce extraskeletal ossification in a murine in vivo implantation assay [3]. COP cells are present in lesions associated with a rare genetic condition, fibrodysplasia ossificans progressiva (FOP), where inflammatory swellings give rise to areas of HO [3]. They are also present in affected heart valve leaflets in end-stage aortic valve disease [4], where vascular injury and inflammation predispose tissue to calcification and ectopic bone formation. These observations suggest that COP cells may home to areas of injury and/or inflammatory and seed early lesions that undergo subsequent ossification. It is unclear if COP cells are broadly associated with NHHO found in various clinical settings. We therefore undertook the current study to provide a systematic evaluation of COP cells as a contributory histopathological finding associated with multiple forms of NHHO.
1. Introduction 2. Materials and methods Non-hereditary heterotopic ossification (NHHO) is the formation of mature bone in non-skeletal tissue, due to multiple injury types. Injuries to the central nervous, musculoskeletal, cutaneous, and cardiovascular systems have been associated with NHHO [1,2]. NHHO may be a clinically severe complication of pelvic and acetabular fractures, as well as following spinal cord, traumatic brain, and other non-neurological injuries.
CD45
Col1
2.1. Patients This study was approved by the Office of Regulatory Affairs institutional review board of the University of Pennsylvania. Thirteen patients underwent orthopaedic evaluation in the University of Pennsylvania
Merge + DAPI
Pre-immune
Trauma
CVA
THA
TBI
SCI
Fig. 1. COP cells are present in lesions of NHHO. Shown are COP cells expressing CD45 and type I collagen (Col1), detected in pre-osseous regions of the indicated NHHO lesions by in situ immunofluorescence. Original magnification is 400×. Red, CD45; Green, Col1; Blue, DAPI. CVA, cerebral vascular accident; THA, total hip arthroplasty; TBI, traumatic brain injury; SCI, spinal cord injury. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: K.P. Egan, et al., Circulating osteogentic precursor cells in non-hereditary heterotopic ossification, Bone (2018), https:// doi.org/10.1016/j.bone.2017.12.028
K.P. Egan et al. / Bone xxx (2018) xxx–xxx
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CD45 Col1 CD45+Col1+
Health System and were diagnosed with NHHO by clinical and radiographic criteria. All patients had decreased range of motion as a result of heterotopic ossification about the involved joint or joints resulting in varying functional limitations. All patients showed evidence of heterotopic bone formation on plain x-rays in varying amounts. Tissue samples of heterotopic bone were divided into the following groups, according to the specific predisposing medical conditions: cerebrovascular accident (CVA), spinal cord injury (SCI), traumatic brain injury (TBI), non-neurologic trauma and post-arthroplasty.
CD45 OCN CD45+OCN+
2.2. Specimen retrieval Bone samples were collected for preparation after resection under routine operating procedures. The diagnosis and setting of NHHO was known in advance of tissue removal and analysis. All specimens were processed within 36 h of harvesting. An average of 5 intact specimens was obtained per individual, ranging in size from 2 mm to 6.4 cm in longest length (before processing).
Fig. 3. COP cells found near blood vessels. COP cells were identified in neovascular regions of a trauma-induced HO lesion by double-labeling immunofluorescence using specific antibodies against either Col1 and CD45 (left panel) or osteocalcin (OCN) and CD45 (right panel). Original magnification is 400 ×. Red, CD45; Green, Col1 or OCN; Blue, DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Tissue samples were fixed in neutral buffered formalin, decalcified, infiltrated and embedded in paraffin, and sectioned at a thickness of 6 μm. Cut samples were then deparaffinized, stained with Harris hematoxylin solution and counterstained with hematoxylin and eosin (H&E) by standard procedures. Other cut tissue sections were stained with Weigert's iron hematoxylin solution and Fast Green stain, and counterstained with Safranin O solution (SAF-O) by standard procedures to confirm the presence of cartilaginous tissue/chondrocytes. All stained slides were examined under light microscopy for histological characteristics of endochondral bone, and for features representative of early and subsequent stages of lesion formation as previously described [5]. At least 30 sections were examined for each specimen and were obtained at multiple levels through the tissue.
washed twice for 5 min in PBS +0.05% Tween-20 (PBST). Secondary antibodies against mouse and rabbit IgGs, tagged with Alexa 555 and Alexa 647 fluorescent labels, respectively, were incubated at a concentration of 1:1000 in a dark humidity chamber for 60 min. After washing twice for 5 min with PBST, 4,6-diamidino-2-phenylindole (DAPI) was incubated with sections for 5 min, followed by a final wash in PBST for 5 min. Sections were viewed with a Nikon Eclipse 90i fluorescent microscope attached to a Photometrics Cool Snap HQ2 camera using NIS-elements Ar version 3.2. Emission times were standardized to the negative control slides. Automated z-stack images were taken and deconvolved to confirm colocalization. Confocal laser scanning microscopy was performed on a Zeiss LSM510 META NLO laser scanning confocal microscope using a Plan-Apo 63 ×/1.4 oil objective. Image capture was performed using Zeiss LSM510 META version 4.2 software (Carl Zeiss Microscopy, Thornwood, NY, USA).
2.4. Immunofluorescence studies
2.5. Quantitation of COP cells in tissue specimens
Heat-induced epitope retrieval at pH 6.0 in 10 mmol/L sodium citrate buffer was performed on all sections. After being heated to 95 °C for 20 min, sections were cooled to room temperature in the buffer. Sections were blocked using 10% serum derived from the same species in which the secondary antibodies were produced. Primary antibodies against CD45 (Santa Cruz Biotechnology) and type 1 collagen (Fitzgerald Industries) or osteocalcin (OCN) (Santa Cruz Biotechnology) were diluted to 1:100 and incubated with sections for 18 h in a dark humidity chamber at 4 °C. For negative technical controls, sections were incubated with normal serum from the same species in which the primary antibodies were produced and at the same concentration as the primary antibodies. Following primary antibody incubation, sections were
Two hundred cells (4,6-diamidino-2-phenylindole [DAPI] positive) were scored in areas of fibroproliferative and neovascular tissue for Col 1+ CD45+ cells. All cells were scored within regions of interest in randomly selected high-power fields (×400).
2.3. Tissue preparation and basic staining
Col1
3. Results 3.1. COP cells are present in lesions of NHHO Samples from 13 patients with NHHO of known precipitating cause were evaluated for the presence of Col1+ CD45+ COP cells (Table 1, Fig. 1). All samples demonstrated the occurrence of COP cells (Fig. 1).
CD45
Merge + DAPI
Fig. 2. COP cells are found in early stage lesions. COP cells were identified in a fibroproliferative region of a trauma-induced HO lesion by double-labeling immunofluorescence using specific antibodies against Col1 and CD45. Original magnification is 400×. Red, CD45; Green, Col1; Blue, DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: K.P. Egan, et al., Circulating osteogentic precursor cells in non-hereditary heterotopic ossification, Bone (2018), https:// doi.org/10.1016/j.bone.2017.12.028
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K.P. Egan et al. / Bone xxx (2018) xxx–xxx
The frequency of COP cells varied from 2 to 14 cells per high power field (Table 1). Lymphoid and brain tissue (unaffected by HO) served as negative (biological) controls for Col1+ CD45+ COP cells (Supplementary Fig. 1). We previously reported that approximately 88% of clonally isolated and expanded OCN+ CD45+ COP cells are Col1 positive [4]. In tissue sections, we found that OCN+ CD45+ COP cells are similarly detected (Supplementary Fig. 2). 3.2. COP cells are found in early stage lesions We recently demonstrated that sequence progression in NHHO lesion formation occurs through six distinct histological stages: (1) perivascular lymphocytic infiltration, (2) lymphocytic migration into soft tissue, (3) reactive fibroproliferation, (4) neovascularity, (5) cartilage formation, and (6) endochondral bone formation [5]. NHHO lesions are histologically mosaic with multiple stages represented depending of their maturity. In order to localize COP cells to stage(s) in the sequence progression, we characterized the histological stages for each type of NHHO (Supplementary Fig. 3) and in consecutive sections attempted to detect Col1+ CD45+ COP cells (Fig. 2 and Table 1). We found that COP cells were localized to early stage lesions of fibroproliferative and neovascular stages (Table 1). OCN+ CD45+ COP cells have a normal reference range of 0.1–3.8% of circulating mononuclear cells in peripheral blood [4,6]. We were able to detect COP cells near blood vessels in the neovascularity stage of lesion formation (Fig. 3). This finding represents the first direct demonstration that COP cells likely migrate to sites of injury and inflammation rather than arising de novo in resident tissue. 4. Discussion This study provides the first systematic evaluation of COP cells as a contributory histopathological finding associated with multiple forms of NHHO. These data support that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites, such as those subject to soft tissue injury, and due to their migratory nature, may likely be involved in seeding sites distant to CNS injury. The presence of COP cells has now been implicated in lesion formation in several forms of trauma-related NHHO (this report), end-stage aortic valvular disease [4], and in FOP [3]. Animal models of ectopic bone formation also support the notion of COP cell involvement in lesion formation [3,7,8]. However a report by Otsuru et al. showed that in a transgenic mouse model CD45+-derived cells do not contribute to mature osteoblasts in BMP-induced HO [9]. Caveats to these findings include species-related differences, and clinical versus BMP-induced inciting events that precipitate HO. For example, Suda et al. reported the presence of donor-derived COP cells in patients that received gender-mismatched hematopoietic stem cell transplantation [3]. Also, local and systemic mediators in human injury-induced NHHO may be vastly different from simple BMP induction in a mouse model, and the former may recruit a repertoire of COP cells from disparate lineages including hematopoietic-derived cells. In an animal model of IL-5 overexpression from hematopoietic-derived cells [10] splenic ossification occurred, suggesting that not only the derivation of the COP cell is important, but also the soluble mediators they secrete as well. Consideration must also be given to the notion that the boneforming function of COP cells may not be their primary role, but rather an adaptive response to injury, repair, or abnormal cytokine signaling [11,12]. The ultimate fate of COP cells may be in tissue regeneration, which under certain conditions dictates de novo bone formation, depending on the microenvironment to which they are drawn [11]. Alternatively, local mesenchymal stem cells may serve as the primary osteochondro-progenitors, while COP cells could
play a role in bone formation at nonskeletal sites and during tissue injury [11]. The presence of COP cells in physiological and pathological processes has been well-established [6,13–15]. Their characterization by cell surface and other markers, possible origins, and migration have also been described [11]. However, prospective identification of COP cells using defined criteria and testing their functionality in well-controlled in vivo assays and studies to identify mechanisms underlying their dual nature as bone-forming and possible bone-regulating cells is still mostly lacking. 5. Conclusions We performed a tissue analysis of COP cells in NHHO secondary to defined conditions, including traumatic brain injury, spinal cord injury, cerebrovascular accident, trauma without neurologic injury, and joint arthroplasty. All specimens revealed the presence of COP cells which were localized to early fibroproliferative and neovascular lesions of NHHO. Supplementary data to this article can be found online at https://doi. org/10.1016/j.bone.2017.12.028. Conflict of interest The authors declare that they have no competing interests. Funding This work was supported by the Ian Cali Distinguished Clinician-Scientist award at the University of Pennsylvania and the Robert and Arlene Professorship in Geriatric Medicine at the Mayo Clinic. Authors' roles Conception and design of the work were by RJP and KPE. Collection and/or assembly of data were by KPE. The manuscript was written by RJP, with revisions by all authors. Data analysis and interpretation were performed by all authors. The manuscript was approved by all authors. References [1] R.J. Pignolo, K.L. Foley, Nonhereditary heterotopic ossification: implications for injury, arthropathy, and aging, Clinical Reviews in Bone and Mineral Metabolism 3 (2005) 261–266. [2] K. Ranganathan, S. Loder, S. Agarwal, V.W. Wong, J. Forsberg, T.A. Davis, S. Wang, A.W. James, B. Levi, Heterotopic ossification: basic-science principles and clinical correlates, J. Bone Joint Surg. Am. 97 (13) (2015) 1101–1111. [3] R.K. Suda, P.C. Billings, K.P. Egan, J.H. Kim, R. McCarrick-Walmsley, D.L. Glaser, D.L. Porter, E.M. Shore, R.J. Pignolo, Circulating osteogenic precursor cells in heterotopic bone formation, Stem Cells 27 (9) (2009) 2209–2219. [4] K.P. Egan, J.H. Kim, E.R. Mohler 3rd, R.J. Pignolo, Role for circulating osteogenic precursor cells in aortic valvular disease, Arterioscler. Thromb. Vasc. Biol. 31 (12) (2011) 2965–2971. [5] K.L. Foley, N. Hebela, M.A. Keenan, R.J. Pignolo, Histopathology of periarticular nonhereditary heterotopic ossification, Bone (2017). [6] P. Gunawardene, A. Al Saedi, L. Singh, S. Bermeo, S. Vogrin, S. Phu, P. Suriyaarachchi, R.J. Pignolo, G. Duque, Age, gender, and percentage of circulating osteoprogenitor (COP) cells: the COP study, Exp. Gerontol. 96 (2017) 68–72. [7] S. Otsuru, K. Tamai, T. Yamazaki, H. Yoshikawa, Y. Kaneda, Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice, Biochem. Biophys. Res. Commun. 354 (2) (2007) 453–458. [8] S. Otsuru, K. Tamai, T. Yamazaki, H. Yoshikawa, Y. Kaneda, Circulating bone marrow-derived osteoblast progenitor cells are recruited to the bone-forming site by the CXCR4/stromal cell-derived factor-1 pathway, Stem Cells 26 (1) (2008) 223–234. [9] S. Otsuru, K.M. Overholt, T.S. Olson, T.J. Hofmann, A.J. Guess, V.M. Velazquez, T. Kaito, M. Dominici, E.M. Horwitz, Hematopoietic derived cells do not contribute to osteogenesis as osteoblasts, Bone 94 (2017) 1–9. [10] M.P. Macias, L.A. Fitzpatrick, I. Brenneise, M.P. McGarry, J.J. Lee, N.A. Lee, Expression of IL-5 alters bone metabolism and induces ossification of the spleen in transgenic mice, J. Clin. Invest. 107 (8) (2001) 949–959.
Please cite this article as: K.P. Egan, et al., Circulating osteogentic precursor cells in non-hereditary heterotopic ossification, Bone (2018), https:// doi.org/10.1016/j.bone.2017.12.028
K.P. Egan et al. / Bone xxx (2018) xxx–xxx [11] R.J. Pignolo, M. Kassem, Circulating osteogenic cells: implications for injury, repair, and regeneration, J. Bone Miner. Res. 26 (8) (2011) 1685–1693. [12] R.J. Pignolo, E.M. Shore, Circulating osteogenic precursor cells, Crit. Rev. Eukaryot. Gene Expr. 20 (2) (2010) 171–180. [13] A. Al Saedi, P. Gunawardene, S. Bermeo, S. Vogrin, D. Boersma, S. Phu, L. Singh, P. Suriyaarachchi, G. Duque, A. Lamin, Expression in circulating osteoprogenitors as a potential biomarker for frailty: the Nepean osteoporosis and frailty (NOF) study, Exp. Gerontol. 96 (2017) 68–72.
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[14] J.J. Alm, H.M. Koivu, T.J. Heino, T.A. Hentunen, S. Laitinen, H.T. Aro, Circulating plastic adherent mesenchymal stem cells in aged hip fracture patients, J. Orthop. Res. 28 (12) (2010) 1634–1642. [15] G.Z. Eghbali-Fatourechi, J. Lamsam, D. Fraser, D. Nagel, B.L. Riggs, S. Khosla, Circulating osteoblast-lineage cells in humans, N. Engl. J. Med. 352 (19) (2005) 1959–1966.
Please cite this article as: K.P. Egan, et al., Circulating osteogentic precursor cells in non-hereditary heterotopic ossification, Bone (2018), https:// doi.org/10.1016/j.bone.2017.12.028
Contents lists available at ScienceDirect
Bone journal homepage: www.elsevier.com/locate/bone
Review Article
Circulating osteogentic precursor cells in non-hereditary heterotopic ossification Kevin P. Egan a, Gustavo Duque b,c, Mary Ann Keenan d, Robert J. Pignolo e,⁎ a
Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States Australian Institute for Musculoskeletal Science (AIMSS), The University of Melbourne and Western Health, Melbourne, VIC, Australia Department of Medicine-Western Health, Melbourne Medical School, The University of Melbourne, Melbourne, VIC, Australia d Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States e Department of Medicine, Mayo Clinic School of Medicine, Mayo Clinic, Rochester, MN, United States b c
a r t i c l e
i n f o
Article history: Received 21 December 2017 Accepted 29 December 2017 Available online xxxx Keywords: Heterotopic ossification Injury Traumatic brain injury Cerebrovascular accident Trauma Spinal cord injury Circulating osteogenic precursor (COP) cells
a b s t r a c t Non-hereditary heterotopic ossification (NHHO) may occur after musculoskeletal trauma, central nervous system (CNS) injury, or surgery. We previously described circulating osteogenic precursor (COP) cells as a bone marrow–derived type 1 collagen+ CD45+ subpopulation of mononuclear adherent cells that are able of producing extraskeletal ossification in a murine in vivo implantation assay. In the current study, we performed a tissue analysis of COP cells in NHHO secondary to defined conditions, including traumatic brain injury, spinal cord injury, cerebrovascular accident, trauma without neurologic injury, and joint arthroplasty. All bone specimens revealed the presence of COP cells at 2–14 cells per high power field. COP cells were localized to early fibroproliferative and neovascular lesions of NHHO with evidence for their circulatory status supported by their presence near blood vessels in examined lesions. This study provides the first systematic evaluation of COP cells as a contributory histopathological finding associated with multiple forms of NHHO. These data support that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites, such as those subject to soft tissue injury, and due to their migratory nature, may likely be involved in seeding sites distant to CNS injury. © 2018 Elsevier Inc. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . 2.1. Patients . . . . . . . . . . . . . . . . . 2.2. Specimen retrieval . . . . . . . . . . . . 2.3. Tissue preparation and basic staining . . . 2.4. Immunofluorescence studies . . . . . . . 2.5. Quantitation of COP cells in tissue specimens 3. Results . . . . . . . . . . . . . . . . . . . . 3.1. COP cells are present in lesions of NHHO . . 3.2. COP cells are found in early stage lesions . . 4. Discussion . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . Funding. . . . . . . . . . . . . . . . . . . . . . . Authors' roles . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .
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⁎ Corresponding author at: Division of Geriatric Medicine & Gerontology, Robert and Arlene Kogod Professor of Geriatric Medicine, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, United States. E-mail addresses: [email protected] (K.P. Egan), [email protected] (G. Duque), [email protected] (R.J. Pignolo).
https://doi.org/10.1016/j.bone.2017.12.028 8756-3282/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: K.P. Egan, et al., Circulating osteogentic precursor cells in non-hereditary heterotopic ossification, Bone (2018), https:// doi.org/10.1016/j.bone.2017.12.028
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K.P. Egan et al. / Bone xxx (2018) xxx–xxx
Table 1 COP cells in early stage NHHO lesions. Diagnosis
Age
Sex
COP cells per HPF (%)
Stage of lesion
TBI
36 40 53 25 42 21 30 38 53 52 80 84 44
M M M M M M M M M M F F F
2 7 6 4 8 6 3 14 2 5 6 2 7
Fibroproliferative Fibroproliferative Fibroproliferative Fibroproliferative Neovascular Fibroproliferative Fibroproliferative Fibroproliferative Fibroproliferative Neovascular Fibroproliferative Fibroproliferative Neovascular
SCI Trauma
Arthroplasty
CVA
HPF, high power field; TBI, traumatic brain injury; SCI, spinal cord injury; CVA, cerebrovascular accident.
Newly formed bone can lead to significant loss of motion at susceptible joints or contribute to stenosis in affected heart valves [1,2]. We previously described circulating osteogenic precursor (COP) cells as a bone marrow–derived type 1 collagen (Col1)+ CD45+ subpopulation of mononuclear adherent cells that are able to produce extraskeletal ossification in a murine in vivo implantation assay [3]. COP cells are present in lesions associated with a rare genetic condition, fibrodysplasia ossificans progressiva (FOP), where inflammatory swellings give rise to areas of HO [3]. They are also present in affected heart valve leaflets in end-stage aortic valve disease [4], where vascular injury and inflammation predispose tissue to calcification and ectopic bone formation. These observations suggest that COP cells may home to areas of injury and/or inflammatory and seed early lesions that undergo subsequent ossification. It is unclear if COP cells are broadly associated with NHHO found in various clinical settings. We therefore undertook the current study to provide a systematic evaluation of COP cells as a contributory histopathological finding associated with multiple forms of NHHO.
1. Introduction 2. Materials and methods Non-hereditary heterotopic ossification (NHHO) is the formation of mature bone in non-skeletal tissue, due to multiple injury types. Injuries to the central nervous, musculoskeletal, cutaneous, and cardiovascular systems have been associated with NHHO [1,2]. NHHO may be a clinically severe complication of pelvic and acetabular fractures, as well as following spinal cord, traumatic brain, and other non-neurological injuries.
CD45
Col1
2.1. Patients This study was approved by the Office of Regulatory Affairs institutional review board of the University of Pennsylvania. Thirteen patients underwent orthopaedic evaluation in the University of Pennsylvania
Merge + DAPI
Pre-immune
Trauma
CVA
THA
TBI
SCI
Fig. 1. COP cells are present in lesions of NHHO. Shown are COP cells expressing CD45 and type I collagen (Col1), detected in pre-osseous regions of the indicated NHHO lesions by in situ immunofluorescence. Original magnification is 400×. Red, CD45; Green, Col1; Blue, DAPI. CVA, cerebral vascular accident; THA, total hip arthroplasty; TBI, traumatic brain injury; SCI, spinal cord injury. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: K.P. Egan, et al., Circulating osteogentic precursor cells in non-hereditary heterotopic ossification, Bone (2018), https:// doi.org/10.1016/j.bone.2017.12.028
K.P. Egan et al. / Bone xxx (2018) xxx–xxx
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CD45 Col1 CD45+Col1+
Health System and were diagnosed with NHHO by clinical and radiographic criteria. All patients had decreased range of motion as a result of heterotopic ossification about the involved joint or joints resulting in varying functional limitations. All patients showed evidence of heterotopic bone formation on plain x-rays in varying amounts. Tissue samples of heterotopic bone were divided into the following groups, according to the specific predisposing medical conditions: cerebrovascular accident (CVA), spinal cord injury (SCI), traumatic brain injury (TBI), non-neurologic trauma and post-arthroplasty.
CD45 OCN CD45+OCN+
2.2. Specimen retrieval Bone samples were collected for preparation after resection under routine operating procedures. The diagnosis and setting of NHHO was known in advance of tissue removal and analysis. All specimens were processed within 36 h of harvesting. An average of 5 intact specimens was obtained per individual, ranging in size from 2 mm to 6.4 cm in longest length (before processing).
Fig. 3. COP cells found near blood vessels. COP cells were identified in neovascular regions of a trauma-induced HO lesion by double-labeling immunofluorescence using specific antibodies against either Col1 and CD45 (left panel) or osteocalcin (OCN) and CD45 (right panel). Original magnification is 400 ×. Red, CD45; Green, Col1 or OCN; Blue, DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Tissue samples were fixed in neutral buffered formalin, decalcified, infiltrated and embedded in paraffin, and sectioned at a thickness of 6 μm. Cut samples were then deparaffinized, stained with Harris hematoxylin solution and counterstained with hematoxylin and eosin (H&E) by standard procedures. Other cut tissue sections were stained with Weigert's iron hematoxylin solution and Fast Green stain, and counterstained with Safranin O solution (SAF-O) by standard procedures to confirm the presence of cartilaginous tissue/chondrocytes. All stained slides were examined under light microscopy for histological characteristics of endochondral bone, and for features representative of early and subsequent stages of lesion formation as previously described [5]. At least 30 sections were examined for each specimen and were obtained at multiple levels through the tissue.
washed twice for 5 min in PBS +0.05% Tween-20 (PBST). Secondary antibodies against mouse and rabbit IgGs, tagged with Alexa 555 and Alexa 647 fluorescent labels, respectively, were incubated at a concentration of 1:1000 in a dark humidity chamber for 60 min. After washing twice for 5 min with PBST, 4,6-diamidino-2-phenylindole (DAPI) was incubated with sections for 5 min, followed by a final wash in PBST for 5 min. Sections were viewed with a Nikon Eclipse 90i fluorescent microscope attached to a Photometrics Cool Snap HQ2 camera using NIS-elements Ar version 3.2. Emission times were standardized to the negative control slides. Automated z-stack images were taken and deconvolved to confirm colocalization. Confocal laser scanning microscopy was performed on a Zeiss LSM510 META NLO laser scanning confocal microscope using a Plan-Apo 63 ×/1.4 oil objective. Image capture was performed using Zeiss LSM510 META version 4.2 software (Carl Zeiss Microscopy, Thornwood, NY, USA).
2.4. Immunofluorescence studies
2.5. Quantitation of COP cells in tissue specimens
Heat-induced epitope retrieval at pH 6.0 in 10 mmol/L sodium citrate buffer was performed on all sections. After being heated to 95 °C for 20 min, sections were cooled to room temperature in the buffer. Sections were blocked using 10% serum derived from the same species in which the secondary antibodies were produced. Primary antibodies against CD45 (Santa Cruz Biotechnology) and type 1 collagen (Fitzgerald Industries) or osteocalcin (OCN) (Santa Cruz Biotechnology) were diluted to 1:100 and incubated with sections for 18 h in a dark humidity chamber at 4 °C. For negative technical controls, sections were incubated with normal serum from the same species in which the primary antibodies were produced and at the same concentration as the primary antibodies. Following primary antibody incubation, sections were
Two hundred cells (4,6-diamidino-2-phenylindole [DAPI] positive) were scored in areas of fibroproliferative and neovascular tissue for Col 1+ CD45+ cells. All cells were scored within regions of interest in randomly selected high-power fields (×400).
2.3. Tissue preparation and basic staining
Col1
3. Results 3.1. COP cells are present in lesions of NHHO Samples from 13 patients with NHHO of known precipitating cause were evaluated for the presence of Col1+ CD45+ COP cells (Table 1, Fig. 1). All samples demonstrated the occurrence of COP cells (Fig. 1).
CD45
Merge + DAPI
Fig. 2. COP cells are found in early stage lesions. COP cells were identified in a fibroproliferative region of a trauma-induced HO lesion by double-labeling immunofluorescence using specific antibodies against Col1 and CD45. Original magnification is 400×. Red, CD45; Green, Col1; Blue, DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: K.P. Egan, et al., Circulating osteogentic precursor cells in non-hereditary heterotopic ossification, Bone (2018), https:// doi.org/10.1016/j.bone.2017.12.028
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K.P. Egan et al. / Bone xxx (2018) xxx–xxx
The frequency of COP cells varied from 2 to 14 cells per high power field (Table 1). Lymphoid and brain tissue (unaffected by HO) served as negative (biological) controls for Col1+ CD45+ COP cells (Supplementary Fig. 1). We previously reported that approximately 88% of clonally isolated and expanded OCN+ CD45+ COP cells are Col1 positive [4]. In tissue sections, we found that OCN+ CD45+ COP cells are similarly detected (Supplementary Fig. 2). 3.2. COP cells are found in early stage lesions We recently demonstrated that sequence progression in NHHO lesion formation occurs through six distinct histological stages: (1) perivascular lymphocytic infiltration, (2) lymphocytic migration into soft tissue, (3) reactive fibroproliferation, (4) neovascularity, (5) cartilage formation, and (6) endochondral bone formation [5]. NHHO lesions are histologically mosaic with multiple stages represented depending of their maturity. In order to localize COP cells to stage(s) in the sequence progression, we characterized the histological stages for each type of NHHO (Supplementary Fig. 3) and in consecutive sections attempted to detect Col1+ CD45+ COP cells (Fig. 2 and Table 1). We found that COP cells were localized to early stage lesions of fibroproliferative and neovascular stages (Table 1). OCN+ CD45+ COP cells have a normal reference range of 0.1–3.8% of circulating mononuclear cells in peripheral blood [4,6]. We were able to detect COP cells near blood vessels in the neovascularity stage of lesion formation (Fig. 3). This finding represents the first direct demonstration that COP cells likely migrate to sites of injury and inflammation rather than arising de novo in resident tissue. 4. Discussion This study provides the first systematic evaluation of COP cells as a contributory histopathological finding associated with multiple forms of NHHO. These data support that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites, such as those subject to soft tissue injury, and due to their migratory nature, may likely be involved in seeding sites distant to CNS injury. The presence of COP cells has now been implicated in lesion formation in several forms of trauma-related NHHO (this report), end-stage aortic valvular disease [4], and in FOP [3]. Animal models of ectopic bone formation also support the notion of COP cell involvement in lesion formation [3,7,8]. However a report by Otsuru et al. showed that in a transgenic mouse model CD45+-derived cells do not contribute to mature osteoblasts in BMP-induced HO [9]. Caveats to these findings include species-related differences, and clinical versus BMP-induced inciting events that precipitate HO. For example, Suda et al. reported the presence of donor-derived COP cells in patients that received gender-mismatched hematopoietic stem cell transplantation [3]. Also, local and systemic mediators in human injury-induced NHHO may be vastly different from simple BMP induction in a mouse model, and the former may recruit a repertoire of COP cells from disparate lineages including hematopoietic-derived cells. In an animal model of IL-5 overexpression from hematopoietic-derived cells [10] splenic ossification occurred, suggesting that not only the derivation of the COP cell is important, but also the soluble mediators they secrete as well. Consideration must also be given to the notion that the boneforming function of COP cells may not be their primary role, but rather an adaptive response to injury, repair, or abnormal cytokine signaling [11,12]. The ultimate fate of COP cells may be in tissue regeneration, which under certain conditions dictates de novo bone formation, depending on the microenvironment to which they are drawn [11]. Alternatively, local mesenchymal stem cells may serve as the primary osteochondro-progenitors, while COP cells could
play a role in bone formation at nonskeletal sites and during tissue injury [11]. The presence of COP cells in physiological and pathological processes has been well-established [6,13–15]. Their characterization by cell surface and other markers, possible origins, and migration have also been described [11]. However, prospective identification of COP cells using defined criteria and testing their functionality in well-controlled in vivo assays and studies to identify mechanisms underlying their dual nature as bone-forming and possible bone-regulating cells is still mostly lacking. 5. Conclusions We performed a tissue analysis of COP cells in NHHO secondary to defined conditions, including traumatic brain injury, spinal cord injury, cerebrovascular accident, trauma without neurologic injury, and joint arthroplasty. All specimens revealed the presence of COP cells which were localized to early fibroproliferative and neovascular lesions of NHHO. Supplementary data to this article can be found online at https://doi. org/10.1016/j.bone.2017.12.028. Conflict of interest The authors declare that they have no competing interests. Funding This work was supported by the Ian Cali Distinguished Clinician-Scientist award at the University of Pennsylvania and the Robert and Arlene Professorship in Geriatric Medicine at the Mayo Clinic. Authors' roles Conception and design of the work were by RJP and KPE. Collection and/or assembly of data were by KPE. The manuscript was written by RJP, with revisions by all authors. Data analysis and interpretation were performed by all authors. The manuscript was approved by all authors. References [1] R.J. Pignolo, K.L. Foley, Nonhereditary heterotopic ossification: implications for injury, arthropathy, and aging, Clinical Reviews in Bone and Mineral Metabolism 3 (2005) 261–266. [2] K. Ranganathan, S. Loder, S. Agarwal, V.W. Wong, J. Forsberg, T.A. Davis, S. Wang, A.W. James, B. Levi, Heterotopic ossification: basic-science principles and clinical correlates, J. Bone Joint Surg. Am. 97 (13) (2015) 1101–1111. [3] R.K. Suda, P.C. Billings, K.P. Egan, J.H. Kim, R. McCarrick-Walmsley, D.L. Glaser, D.L. Porter, E.M. Shore, R.J. Pignolo, Circulating osteogenic precursor cells in heterotopic bone formation, Stem Cells 27 (9) (2009) 2209–2219. [4] K.P. Egan, J.H. Kim, E.R. Mohler 3rd, R.J. Pignolo, Role for circulating osteogenic precursor cells in aortic valvular disease, Arterioscler. Thromb. Vasc. Biol. 31 (12) (2011) 2965–2971. [5] K.L. Foley, N. Hebela, M.A. Keenan, R.J. Pignolo, Histopathology of periarticular nonhereditary heterotopic ossification, Bone (2017). [6] P. Gunawardene, A. Al Saedi, L. Singh, S. Bermeo, S. Vogrin, S. Phu, P. Suriyaarachchi, R.J. Pignolo, G. Duque, Age, gender, and percentage of circulating osteoprogenitor (COP) cells: the COP study, Exp. Gerontol. 96 (2017) 68–72. [7] S. Otsuru, K. Tamai, T. Yamazaki, H. Yoshikawa, Y. Kaneda, Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice, Biochem. Biophys. Res. Commun. 354 (2) (2007) 453–458. [8] S. Otsuru, K. Tamai, T. Yamazaki, H. Yoshikawa, Y. Kaneda, Circulating bone marrow-derived osteoblast progenitor cells are recruited to the bone-forming site by the CXCR4/stromal cell-derived factor-1 pathway, Stem Cells 26 (1) (2008) 223–234. [9] S. Otsuru, K.M. Overholt, T.S. Olson, T.J. Hofmann, A.J. Guess, V.M. Velazquez, T. Kaito, M. Dominici, E.M. Horwitz, Hematopoietic derived cells do not contribute to osteogenesis as osteoblasts, Bone 94 (2017) 1–9. [10] M.P. Macias, L.A. Fitzpatrick, I. Brenneise, M.P. McGarry, J.J. Lee, N.A. Lee, Expression of IL-5 alters bone metabolism and induces ossification of the spleen in transgenic mice, J. Clin. Invest. 107 (8) (2001) 949–959.
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Please cite this article as: K.P. Egan, et al., Circulating osteogentic precursor cells in non-hereditary heterotopic ossification, Bone (2018), https:// doi.org/10.1016/j.bone.2017.12.028