Introduction

Osteoprogenitor cells, also known as osteogenic cells, are stem cells in the bone that play a prodigal role in bone repair and growth.[1] These cells are the precursors to the more specialized bone cells (osteocytes and osteoblasts) and reside in the bone marrow. Osteoprogenitor cells originate from infant mesenchymal cells and turn into spindle cells at the surface of matured bones. In developing bones, they appear more frequently and activate multifunctional stages to remodel the bones. The body loses the ability to synthesize or utilize more osteoprogenitor cells with age. Dysfunction of osteoprogenitor cells may delay ossification and lead to a spectrum of diseases such as dwarfism and Kashin-Beck disease.[2]

Structure

Osteoprogenitor cells are often referred to as preosteoblasts. They can be present within the endosteum, the cellular layer of the periosteum, and the lining of the osteogenic cells. In matured bones that no longer display active bone remodeling or formation, osteoprogenitor cells exist as flattened spindle-shaped structures. They attach to the bone surface and are referred to as inactive osteoblasts during this period. In maturing bones, however, these cells appear in their largest form. During fetal development or high turnover periods in adult osteogenesis, numerous osteoprogenitor cells function to give rise to osteoblasts. These structures display plump oval nuclei and emboldened abundant spindle-shaped cytoplasm at this stage, later converting to characteristic cuboidal active osteoblasts.[3][4]

Function

Osteoprogenitors can self-proliferate and self-renew. They participate in osteogenic differentiation and play a role in regulating angiogenesis.[5] Osteoprogenitor cells can differentiate into osteoblasts through mitotic division or by dividing into two stem cells via a highly regulated mechanism, which remains static during the proliferation process. After completing DNA synthesis and cell expansion, the cell retains its original genetic information. The endocrine system and local factors (growth factors and cytokines) mainly regulate osteogenesis.

Neuropeptide Y is a potent regulator of osteogenesis, and recent studies have confirmed that osteoprogenitor cells originating from hypothalamic Y2-/- mice have enhanced osteogenic activities.[6] Proline-rich tyrosine kinase 2 supposedly regulates the differentiation of early osteoprogenitors, and proline-rich tyrosine kinase 2 inhibitors promote osteogenesis and could act as a treatment for osteoporosis.[7] Osteoprogenitor cells also reside in the perichondrium. These osteoprogenitors hyper-regulate bone morphogenetic proteins during differentiation into mature osteoblasts responsible for the production of bone matrix.[8]

Tissue Preparation

Bone marrow stromal cells (BMSCs) yield testable osteoprogenitors. The cell samples are diluted with phosphate-buffered saline (PBS) at a ratio of 1:3 then nucleated cells are isolated with a density gradient solution. Preparatory phase complete medium (CM) consists of  0.1 mmol/L nonessential amino acids, alpha-MEM with 10% fetal bovine serum, 4.5 mg/mL d-glucose, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 mmol/L HEPES buffer, 100 μg/mL streptomycin, and 0.29 mg/mL l-glutamine.[9] 

The collected nucleated cells are plated at a density of 100,000 cells/cm^2 in supplemented CM. The CM supplement requires an additional 5 ng/mL fibroblast growth factor-2 (GF2) and 10 nmol/L dexamethasones; both elements proliferate osteogenic commitment of bone marrow stromal cells. The supplemental process undergoes humidification at 37 degrees C in a 5% CO2 incubator.[10]

Histochemistry and Cytochemistry

The development of osteoprogenitor cells has been studied by several research groups. A study suggests that the perichondrial Thy-1–positive cells demonstrate potential osteogenic activity and participate in osteoblast formation during endochondral ossification. Another study shows intense alkaline phosphatase activity two weeks after osteogenic induction and the presence of mineralized nodules. The expression of bone sialoprotein, dentin matrix protein-1, and osteocalcin increases due to ALP proliferation. 55 to 65% of cells, as confirmed by flow cytometry, display the cell-surface markers Sca1+ and Thy1+ in vitro expansion for an alpha-SMA-GFP positive population. The alpha-SMA–GFP-positive population also exhibits high proliferative and osteogenic probabilities when compared to an alpha-SMA–GFP-negative population.[8][11]

Microscopy, Light

Final visualization of osteoprogenitor cells under the microscope involves a delicate preparation phase: the explants are fixed in 4% buffered formalin for one whole day, and samples are decalcified with 0.5 mol/L ethylenediaminetetraacetic acid (pH 8) for 7 to 10 days. The samples are deposited in paraffin, and the cross-sections of the samples are prepared with 5 um thickness at three different strata.[12]

The samples are stained with hematoxylin/eosin and Masson/Trichrome, and both a qualitative exam for the presence of bone tissue and a quantitative exam by computerized bone histomorphometry is conducted. For each hematoxylin/eosin-stained cross-section, 3 or 4 images (adequate to fill the construct cross-sections) are required to calculate the bone tissue area and available area for tissue ingrowth (net implant area - undegraded scaffold area) by digital imaging analysis. Under the visuals of scanning electron microscopy (SEM), the red color in the slides that are stained by Masson/Trichrome indicates lamellar and remodeled bone, while the blue color shows freshly deposited and immature bone.[9]

Pathophysiology

Bone tumors are formed within osteoprogenitor or stromal cell lineage during Paget disease (PD), which likely derive from genetic alterations related to familial Paget’s disease. During Paget’s disease, the endosteal surface undergoes active remodeling, and abnormal osteoclasts bearing nuclear inclusions occur. The fibrotic tissues in bone biopsies from PD patients are considered to be made of surplus elongated, sophisticatedly branched stromal cells that display high alkaline phosphatase levels; these cells are similar to the pre-osteogenic stromal cells located within the normal bone marrow.[13]

Early and full-blown pagetic lesions show dynamic transformations in the arrangement, number, and function of stromal cells within endosteal/medullary tissue. An excessive amount of bone marrow osteoprogenitor cells in pagetic lesion areas verify earlier data on static and dynamic histomorphometry in patients with Paget’s disease. Previous data revealed that the rate of osteogenesis increases during Paget disease, but research has confirmed that there is also a surge in the birthrate of osteoblasts.[14]

The quality of the new bones depends on nature and hypermineralization taking place. Most stromal/osteoblastic abnormalities result from bone mass increment, characterized by denser and thicker trabecular structures. Compromised mechanical integrity caused by poor architectural organization and transformations within the mineralized matrix material is an additional characteristic.[15]

Clinical Significance

Mesenchymal stem cells (MSCs) collected from adipose and bone marrow tissue hold therapeutic value for various bone disease treatments. Current studies demonstrate the benefit of bone grafts based on combinations of MSC, biomimetic scaffolds, and growth factor delivery, which showed an increased osteogenic regeneration rate with minimal side effects. The specific mechanisms of cellular signaling in bone remodeling are essential in understanding the incorporation of newer effective treatment methods for numerous bone diseases.[16]

Various transplant therapies involving osteogenic autologous bone grafts are less frequently used; patient-specific cell therapies involving autologous BM-MNCs (bone marrow mononuclear cells) composed of stem cells, monocytes, lymphocytes, and dendritic cells are rising in popularity for bone pathology treatments. All patients suffering from long-bone pseudoarthrosis attained full bone consolidation when treated with autologous BM-MNCs and allogeneic cancellous bone grafts. Bone marrow aspirate utilized during osteonecrosis treatment via minimally invasive decompression of the femoral head decreased disease progress and yielded overall pain and symptom relief. Bone marrow concentrated injections lead to better osteogenic unions, resulting in complete recovery in post-operative achondroplastic dwarf patients within 2 to 10 months after femoral lightning surgeries. Intravenous BMC injections, mixed with iloprost, proliferate fracture healing in patients with avascular necrosis. Patients receiving Intra-articular BMNc displayed better chewing and maximum interincisal opening with integral pain relief.[17][16]