Tissue engineering 1: Autologous transplantation of peripheral blood CD34⁺ cells in patients with femoral and tibial fracture non-union

Author affiliations: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.

About 5–10% of bone fractures fail to heal after conventional treatment. Stem cell therapy is promising for treating these cases, but initial results using mesenchymal stem cells have been disappointing. In a phase I/II clinical study, researchers associated with the Translational Research Center for Medical Innovation (TRI) in Japan have shown that transplantation of autologous CD34⁺ cells from peripheral blood in patients with non-union fractures is safe and effective way to heal such non-union fractures.

1. INTRODUCTION

Recent years have seen remarkable developments in the field of regenerative medicine as a result of research into embryonic stem cells and induced pluripotent stem cells. However, unresolved issues with the use of these so-called pluripotent cells, including ethical concerns and the potential for neoplastic cell transforma­tion, make their clinical application problematic. Research into organogenesis and tissue regeneration has hence mainly focused on somatic stem cells. Regeneration of bone and cartilage using mesenchymal stem cells derived from the bone marrow or the synovial membrane is now attracting attention in orthopaedics and has already been used clinically. However, serious disadvantages remain, including the invasiveness of bone marrow collection, the difficulty and slowness of cell culture techniques, and the limited effectiveness of therapy. Thus, less invasive and more effective therapies are needed.

Vascular research has been conducted to establish the pathogenesis and improve the treatment of vascular diseases. Developments up until now in this field have not always been comparable with the most advanced research in fields such as oncology, hematology and neurology. However, the recent detailed characteriza­tion of the differentiation mechanisms involved in vascularization has opened up a new field of vascular re­search, incorporating trends in fields such as hematology and embryology. In the field of regenerative med­icine, which is now part of mainstream medicine, research into organogenesis and tissue regeneration using various stem cells is underway, but many aspects of the mechanisms by which the vascular system can be re­constructed to nourish regenerated organs and tissues remain unclear. Vasculogenesis thus has the potential to play a key role in the development of regenerative medicine. After Asahara et al. discovered human periph­eral blood vascular endothelial progenitor cells (EPCs) in 1997¹, several studies have elucidated the mecha­nism of vasculogenesis involving EPCs, in addition to that of conventional angiogenesis involving pre-exist­ing vascular endothelial cells^2,3. Revascularization is currently performed as therapy for lower-limb ischemia and ischemic cardiac diseases. The significance of vascularization, particularly for bone regeneration, has long been recognized in orthopaedics.

This article presents an overview of basic research, non-clinical studies and a phase I/II clinical study to investigate the in vivo kinetics of EPCs/peripheral blood CD34⁺ cells and bone revascularization therapy us­ing EPCs/peripheral blood CD34⁺ cells, which have great potential for fracture treatment.

2. ANGIOGENESIS AND VASCULOGENESIS

Moses Judah Folkman first established the concept of angiogenic growth factor and its application to ther­apy, and he showed that angiogenic growth factors were expressed as a result of cancer growth and infiltra­tion. He defined angiogenesis as the process of forming new blood vessels from pre-existing adjacent blood vessels by the proliferation and migration of vascular endothelial cells induced by growth factors⁴. The re­cent discovery of various growth factors, including vascular endothelial growth factor (VEGF), has led to the clinical application of therapy that promotes angiogenesis. However, some patients derived no clear benefit from this therapy. In these patients, the response of pre-existing vascular endothelial cells to vascular growth factors was probably lower at the ischemic lesion site. Therefore, the next challenge was to deliver highly re­sponsive vascular endothelial cells to ischemic lesions.

In 1997, Asahara et al. reported that EPCs were present in adult blood circulation and involved in vascu­larization in severe ischemic lesions¹. This mechanism was found to be con­sistent with vasculogenesis, a process previously thought to occur only dur­ing fetal life, in which undifferentiated vascular EPCs migrate to a local site, where they reside, proliferate and differentiate to form a new blood vessel. This discovery led to the development of a concept distinct from angiogen­esis, which is the conventional concept of vascularization in adults, where vascular endothelial cells proliferate and migrate to adjacent pre-existing blood vessels. When ischemia, cancer, wound healing or neovascularization in the uterus and the ovary were induced in a transgenic mouse model of bone-marrow transplantation that expressed β-galactosidase following ex­pression of vascular endothelial cell-specific genes (Flk-1, Tie-2), new blood vessels formed by cells expressing donor bone marrow–derived Flk-1 or Tie- 2^5,6. These studies showed that vascularization is achieved by both vasculo­genesis and angiogenesis in adults as well as in fetal life. Neovascularization in adults is now thought to be mediated by an interaction between angio­genesis and vasculogenesis.

3. FRACTURE HEALING BY PERIPHERAL BLOOD EPCS/CD34⁺ CELLS

Bone fractures usually heal through anatomical reduction and rigid im­mobilization. However, bone union fails to occur (non-union fractures) in 5–10% of patients with fractures, partly because appropriate circulation is not achieved at the fracture site. Autologous free cancellous bone grafting is performed and may be followed by vascularized bone grafting with a fo­cus on revascularization, but healing of a non-union fracture is often diffi­cult. We therefore focused on neovascularization through cell therapy using EPCs and conducted basic experiments to examine the usefulness of this therapy for non-union fractures in preclinical animal studies.

In a preliminary study, we analysed the in vivo kinetics of EPCs in the fracture healing process by developing a mouse model of closed fracture and using fluorescence-activated cell sorting. EPCs were found to increase in the bone marrow and the peripheral blood after fracture. Subsequently, to demonstrate local differentiation of bone marrow–derived EPCs into vascu­lar endothelial cells, double immunostaining of Sca1 and CD31 in a Tie2/ LacZ transgenic mouse model of bone marrow transplantation was per­formed. It showed that bone marrow–derived EPCs differentiated into vas­cular endothelial cells at the fracture site. These results demonstrated that fracture-induced mobilization of EPCs from the bone marrow to the pe­ripheral blood occurs and that EPCs contributed to healing at the fracture site⁷. Other research groups have also reported that EPCs were mobilized to the fracture site in humans⁸ and to the distraction osteogenesis site in a rat distraction osteogenesis model⁹. These reports support our findings.

Given that revascularization using peripheral blood cells was possible, it was logical to assume that differentiation of human peripheral blood CD34⁺ cells into vascular endothelial cells would promote bone regeneration. In our next study, we therefore intravenously transplanted 10⁵ CD34⁺ cells col­lected from the peripheral blood of healthy humans into a nude rat mod­el of non-union fracture and demonstrated that these cells accumulated at the non-union site and contributed to fracture healing by inducing an envi­ronment suitable for healing through both vasculogenesis and osteogenesis (Fig. 1)¹⁰. Radiological and histological assessments showed that non-union was still present in all animals in the control groups (one control group in­jected with phosphate-buffered saline [PBS] and one transplanted with hu­man mononuclear cells) 8 weeks postoperatively, whereas bone union was achieved in 66% of animals at 4 weeks and in all animals at 8 weeks in the CD34⁺ cell transplant group (Table 1A). Mechanical assessment by a three-point bending test demonstrated significantly stronger fracture healing in the CD34⁺ cell transplant group than in the control groups.

Fluorescently labeled CD34⁺ cells were found to accumulate preferen­tially at the non-union site and contribute to revascularization and bone regeneration in collaboration with rat-derived endothelial cells and osteo­blasts. Two weeks after transplantation, histological and molecular biolog­ical analyses showed the presence of human-derived mature vascular en­dothelial cells and osteoblasts at the non-union site, demonstrating that CD34⁺ cells had differentiated into mature endothelial cells and osteoblasts. Single-cell reverse transcriptase polymerase chain reaction (RT-PCR) analy­sis showed that approximately 20% of CD34⁺ cells co-expressed osteocalcin, an osteoblast-specific marker, immediately after isolation, and that human CD34⁺ cells appeared to directly differentiate into osteoblasts without fus­ing with rat cells. Determination of the microvessel density and the osteo­blast density in rats by immunostaining at the non-union site 2 weeks after transplantation showed that angiogenesis and osteogenesis were significant­ly increased in the CD34⁺ cell transplant group compared with both control groups. Because RT-PCR analysis identified angiogenic factors such as hu­man-derived VEGF at the non-union site, the CD34⁺ cells were considered to play a paracrine role locally. Administration of soluble Flt1, a VEGF an­tagonist, inhibited not only angiogenesis but also osteogenesis, showing that angiogenesis by CD34⁺ cells plays a key role in the fracture healing process.

With eventual clinical application in mind, we also conducted an ex­periment to confirm that locally transplanted human peripheral blood CD34⁺ cells induced an environment suitable for fracture healing at the non-union site through both vasculogenesis and osteogenesis and contrib­uted to fracture healing in a dose-dependent manner¹¹. First, peripheral blood granulocyte colony stimulating factor (G-CSF)-mobilized CD34⁺ cells were transplanted locally to the non-union site of the same animal model (using atelocollagen as a carrier). The numbers of cells transplant­ed were based on the results of the above-described intravenous transplan­tation. Radiological and histological assessments showed that bone union was achieved in 33% of animals at 4 weeks and all animals at 8 weeks af­ter transplantation of 10⁵ cells, and in 11% of animals at 4 weeks and 33% at 8 weeks after transplantation of cells, but not after transplantation of 10³ cells or in PBS controls (Table 1B). Determination of the microvessel density and the osteoblast density at the non-union site by immunostain­ing at 2 weeks showed dose-dependent increases in angiogenesis and os­teogenesis. Greater levels of angiogenesis and osteogenesis were observed after transplantation of 10⁴ cells and 10⁵ cells than after transplantation of 10³ cells or PBS injection. These results suggest that local transplantation of peripheral blood CD34⁺ cells would be useful as a new treatment op­tion for non-union fractures.

To confirm the superiority of human peripheral blood CD34⁺ cells over mononuclear cells, we transplanted both cell types locally to the non-union site of the nude rat non-union fracture model and compared their effects. Transplantation of 10⁷ mononuclear cells induced an environment suitable for fracture healing through both vasculogenesis and osteogenesis and con­tributed to fracture healing, but the efficacy was significantly inferior to that of transplantation of 10⁵ CD34⁺ cells as measured by microvessel den­sity, osteoblast density, and the density of human-derived mature vascu­lar endothelial cells and osteoblasts, as well as by radiological, histological and mechanical assessments¹². These effects of CD34⁺ and mononuclear cell transplantation have been confirmed not only in this refractory non-union fracture model but also in the established non-union model¹³. The re­sults were attributed partly to infiltration of more inflammatory cells in the mononuclear cell transplant group than in the CD34⁺ cell transplant group.

Further characterization of the mechanisms involved in fracture healing associated with EPCs would lead to more improvement in therapeutic ef­fects. We therefore investigated the healing effects of mobilization of EPCs to the non-union site by sustained released G-CSF¹⁴ and simvastatin¹⁵, the healing effect of extracellular mobilization of adaptor protein Lnk^16,17 medi­ated by SCF/c-Kit signalling, and mobilization of EPCs mediated by SDF- 1/CXCR4 signalling¹⁸. All studies confirmed that EPCs are closely associat­ed with fracture healing.

4. CLINICAL STUDY: BONE REVASCULARIZATION BY TRANS­PLANTATION OF PERIPHERAL BLOOD CD34⁺ CELLS

Based on the above-described non-clinical research results, a phase I/II clin­ical trial of bone revascularization by transplantation of autologous periph­eral blood CD34⁺ cells was conducted in patients with non-infectious, non-union fractures, following approval by the Review Committee on Clinical Research of Human Stem Cells on 4 September 2009. An advantage of bone revascularization therapy by transplantation of autologous peripher­al blood CD34⁺ cells over conventional therapies is that it regenerates both bone and new blood vessels after proliferation of cells in an undifferentiated state and subsequent differentiation at the non-union site.

Eligible patients (two with femoral non-union fractures and five with tibial non-union fractures) aged between 20 and 70 years provided writ­ten informed consent in person. After obtaining consent, screening tests, a case review meeting and an eligibility assessment were conducted and pa­tients were then enrolled. CD34⁺ cell isolation, non-union surgery and cell transplantation were performed at the Institute of Biomedical Research and Innovation, and patients were then transferred to Kobe University Hospi­tal for observation.

G-CSF was subcutaneously injected for 5 days to mobilize CD34⁺ cells into the peripheral blood. Mononuclear cells were collected by apheresis, and CD34⁺ cells were isolated by magnetic-activated cell sort­ing. Treatment involved non-union surgery (to improve internal fixation as needed in addition to autologous bone grafting) on day 6 and trans­plantation of 5 × 10⁵ cells kg⁻¹ of autologous peripheral blood CD34⁺ cells using atelocollagen as a carrier (Fig. 2). The primary endpoints were safety and the presence or absence of radiological fracture healing 12 weeks after surgery.

We first reported safety and efficacy in a patient who had tibial non-union¹⁹. The mean postoperative time to clinical bone union was 12.6 weeks, and the mean postoperative time to radiological bone union was 16.1 weeks (Table 2)²⁰. The efficacy of the therapy was demonstrated — 71.4% of patients achieved radiological bone union at 12 weeks after sur­gery, the primary endpoint, compared with a healing rate of 18.1% in 11 patients with femoral or tibial non-union who underwent concomitant bone grafting in the same medical institution, a patient group selected as a historical control (Table 3)²⁰. The safety evaluation 1 year after surgery showed no serious adverse events.

5. FUTURE PROSPECTS

The significance and efficacy of EPCs/CD34⁺ cells in fracture healing de­scribed so far have attracted the attention of other research groups in re­cent years, and the results of basic research continue to accumulate. The effect of mobilization of EPCs to the peripheral blood has been reported for the rat model of tibial distraction osteogenesis²¹ and the mouse model of femoral fracture²² as well as for tibial long bone fracture in humans^8,9. As well as in our immunodeficient rat non-union fracture model^10–12, im­munodeficient rat non-union model¹³, and rat femoral defect model^23,24, the efficacy of EPCs/CD34⁺ cells in the fracture healing process has also been reported for the ovine tibial defect model²⁵. Thus, the importance of EPCs/CD34⁺ cells has been increasingly recognized in recent years^26–29.

The phase I/II clinical study demonstrated the efficacy and safety of this therapy in patients with non-union fractures. With the aim of establishing this therapy as a standard of care, we need to conduct a multicentre, inves­tigator-initiated study in a larger number of patients. Transplantation of pe­ripheral blood CD34⁺ cells, a novel therapy for non-union fractures, has al­ready demonstrated high efficacy and safety in animal experiments and early clinical studies, and excellent therapeutic effects are expected in future ex­tensive clinical applications.

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