Targeting the cannabinoid system for pain relief?

Marijuana has been used to relieve pain for centuries, but its analgesic mechanism has only been understood during the past two decades. It is mainly mediated by its constituents, cannabinoids, through activating central cannabinoid 1 (CB₁) receptors, as well as peripheral CB₁ and CB₂ receptors. CB₂-selective agonists have the benefit of lacking CB₁ receptor-mediated CNS side effects. Anandamide and 2-arachidonoylglycerol (2-AG) are two intensively studied endogenous lipid ligands of cannabinoid receptors, termed endocannabinoids, which are synthesized on demand and rapidly degraded. Thus, inhibitors of their degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase (MAGL), respectively, may be superior to direct cannabinoid receptor ligands as a promising strategy for pain relief. In addition to the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosynthesis and degradation processes, we also review recent studies that revealed a novel analgesic mechanism, involving 2-AG in the periaqueductal gray (PAG), a midbrain region for initiating descending pain inhibition. It is initiated by Gq-protein-coupled receptor (GqPCR) activation of the phospholipase C (PLC)-diacylglycerol lipase (DAGL) enzymatic cascade, generating 2-AG that produces inhibition of GABAergic transmission (disinhibition) in the PAG, thereby leading to analgesia. This GqPCR-PLC-DAGL-2-AG retrograde disinhibition mechanism in the PAG can be initiated by activating type 5 metabotropic glutamate receptor (mGluR5), muscarinic acetylcholine (M1/M3), and orexin (OX1) receptors. mGluR5-mediated disinhibition can be initiated by glutamate transporter inhibitors, or indirectly by substance P, neurotensin, cholecystokinin, capsaicin, and AM404, the bioactive metabolite of acetaminophen in the brain. The putative role of 2-AG generated after activating the above neurotransmitter receptors in stress-induced analgesia is also discussed.     

Cannabinoids and Bone: Friend or Foe?

The endocannabinoid system is recognized to play an important role in regulating a variety of physiological processes, including appetite control and energy balance, pain perception, and immune responses. The endocannabinoid system has also recently been implicated in the regulation of bone metabolism. Endogenously produced cannabinoids are hydrophobic molecules derived from hydrolysis of membrane phospholipids. These substances, along with plant-derived and synthetic cannabinoids, interact with the type 1 (CB₁) and 2 (CB₂) cannabinoid receptors and the GPR55 receptor to regulate cellular function through a variety of signaling pathways. Endocannabinoids are produced in bone, but the mechanisms that regulate their production are unclear. Skeletal phenotyping of mice with targeted inactivation of cannabinoid receptors and pharmacological studies have shown that cannabinoids play a key role in the regulation of bone metabolism. Mice with CB₁ deficiency have high peak bone mass as a result of an osteoclast defect but develop age-related osteoporosis as a result of impaired bone formation and accumulation of bone marrow fat. Mice with CB₂ deficiency have relatively normal peak bone mass but develop age-related osteoporosis as a result of increased bone turnover with uncoupling of bone resorption from bone formation. Mice with GPR55 deficiency have increased bone mass as a result of a defect in the resorptive activity of osteoclasts, but bone formation is unaffected. Cannabinoids are also produced within synovial tissues, and preclinical studies have shown that cannabinoid receptor ligands are effective in the treatment of inflammatory arthritis. These data indicate that cannabinoid receptors and the enzymes responsible for ligand synthesis and breakdown play important roles in bone remodeling and in the pathogenesis of joint disease.     

A Cost-Effective Method for the Automatic Quantitative Analysis of Fibroblastic Colony-Forming Units

A great deal of the work characterizing stromal cell precursors in the bone marrow has been performed using the fibroblastic colony-forming unit (CFU-f) assay. However, the assay is limited in its usefulness by the necessity for manual colony counting which means that assay quantitation is highly subjective, time consuming, and much information regarding the colony size is lost. To rectify this, we have developed a computer-automated method for the analysis of CFU-f. Bone marrow cells were cultured at low density and treated with either prostaglandin E₂ (PGE₂), basic fibroblast growth factor (bFGF), or dexamethasone, and colony formation was assessed by staining with methylene blue. After staining, the dishes were photographed over a light box using a digital camera and the image was then analyzed using Bioimage ``Intelligent Quantifier' image analysis software which automatically locates and quantifies each individual colony. The data can then be imported to a spreadsheet program and processed. We have shown that this system can accurately identify, assign coordinates, and quantitate each individual colony. Colony numbers obtained with this method and manually counting showed a linear relationship with a correlation coefficient of 0.99. In addition, using the colony intensity and surface area data, the colony size can be calculated. With this methodology, we have shown that dexamethasone, PGE₂, and bFGF can all modulate total cell numbers in bone marrow stromal cells (BMSC) cultures but modulating both colony number and colony size. Received: 22 January 1998 / Accepted: 10 January 1999     

The role of glucocorticoids and prostaglandin E2 in the recruitment of bone marrow mesenchymal cells to the osteoblastic lineage: Positive and negative effects

The role of glucocorticoids in bone formation presents a problem because although pharmacological doses in vivo give rise to osteoporosis, physiological concentrations are required for osteoblast (OB) differentiation in vitro. To try and rationalize this dichotomy, we investigated the effect of dexamethasone on the recruitment of OB precursors present in bone marrow. Using the CFU-f assay, we can measure (1) total colony formation; (2) the osteoblastic differentiation of the colonies defined as their ability to express alkaline phosphatase, synthesize collagen, and to calcify; and (3) colony expansion as either average colony surface area or average colony number. In control cultures and in the presence of 10⁻¹⁰–10⁻⁹ M dexamethasone, colony formation and total cell number was maximal, but the addition of PGE₂ had no effect on colony number and very few colonies expressed the OB phenotype. In the presence of 10⁻⁸–10⁻⁷ M dexamethasone, colony numbers and total cell numbers were reduced but were increased by the addition of PGE₂, the average colony cell number and surface area were relatively unchanged and a proportion of the colonies expressed APase, calcified and synthesized collagen. In cultures containing 10⁻⁶–10⁻⁵ M dexamethasone, colony numbers were further reduced but were stimulated by the addition of PGE₂ and some colonies differentiated; however, colony expansion was dramatically reduced by up to 80%. These results suggest that physiological levels of glucocorticoids are necessary for OB differentiation and allow the control of OB recruitment by PGE₂. High levels of glucocorticoids drastically reduce proliferation of the OB precursors leading to glucocorticoid-induced osteoporosis. Received: 8 May 1995 / Accepted: 23 February 1996     

Fibrillin‐1 Regulates Skeletal Stem Cell Differentiation by Modulating TGFβ Activity Within the Marrow Niche

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age‐related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFβ modulator fibrillin‐1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin‐1 (Fbn1^Prx1–/– mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1^Prx1–/– mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1^Prx1–/– mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1‐silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin‐1 modulation of TGFβ bioavailability, cultures of marrow stromal cells from Fbn1^Prx1–/– limb bones showed improper overactivation of latent TGFβ. In line with this finding, systemic TGFβ neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin‐1 regulates MSC activity by modulating TGFβ bioavailability within the microenvironment of marrow niches. © 2015 American Society for Bone and Mineral Research.     

Centrifugal Isolation of Bone Marrow from Bone: An Improved Method for the Recovery and Quantitation of Bone Marrow Osteoprogenitor Cells from Rat Tibiae and Femurae

SUMMARY. The high variation often observed in the ex vivo fibroblastic-colony forming unit (CFU-f) assay is likely to be due to both biological and experimental variation. To determine whether we could improve experimental methods we developed an alternative method of bone marrow cell (BMC) isolation employing a centrifugation step. The osteogenic capacity of centrifugally isolated BMC was compared to that of BMC that were isolated using the standard ``flushing' technique using the CFU-f assay. The centrifugation method was found to be both quick and simple to perform and allowed simultaneous preparation of all samples. Centrifugually isolated BMC gave rise to approximately 100% more cfu-ap and cfu-f in cultures from both tibiae and femurae. The proportion of alkaline phosphatase positive colonies remained the same and colony morphologies were similar for both isolation methods. Histological comparison of the flushed and spun bones showed that after the flushing procedure many cells remained in the marrow cavity especially in the trabecular area. In contrast, centrifugation completely emptied the marrow space of all cells except bone lining cells and osteoblasts. Thus the osteogenic capacity of the bone marrow can be expressed as the number of CFU-f per bone instead of the frequency as is the norm. Using these methods to isolate BMC for ex vivo investigations should lead to a reduction in CFU-f number variation due to the isolation method. Received: 7 October 1998 / Accepted: 26 July 1999     

Number and Proliferative Capacity of Human Mesenchymal Stem Cells Are Modulated Positively in Multiple Trauma Patients and Negatively in Atrophic Nonunions

Mesenchymal stem cells (MSCs) participate in regenerative osteogenesis by generating bone-forming cells. To examine the proliferative capacity of MSC populations from bone marrow and their relationship to trauma severity (multiple trauma, monofracture, atrophic nonunion), we quantified colony properties of human MSCs in vitro. Serum levels of mediators associated with bone formation were also assessed. Fifty-five individuals were enrolled in this study (13 multiple trauma patients, 15 patients with monofracture, 20 patients with atrophic nonunions, 7 healthy volunteers). The colony forming unit-fibroblast (CFU-F) assay was used to quantify total colony number, mean cell density per colony, and mean colony area. MSC phenotype was established using flow cytometry and osteogenic differentiation. MSCs obtained from multiple-trauma patients yielded the highest reservoir. Significant differences in colony numbers of MSCs in female subjects were found between multiple-trauma patients (mean ± SD 48 ± 21 CFU-F/culture) and healthy volunteers (18.7 ± 3.3 CFU-F/culture, P < 0.05), patients with monotrauma (15 ± 10 CFU-F/culture, P < 0.05), and patients with atrophic nonunions (6.3 ± 4.1 CFU-F/culture, P < 0.05). In male participants, significant differences were found between patients with nonunions (14 ± 14 CFU-F/culture) and healthy volunteers (54 ± 17 CFU-F/culture, P < 0.05) as well as multiple-trauma patients (59 ± 25 CFU-F/culture, P < 0.05). The highest proliferative capacity (cell density) was seen in multiple-trauma patients. These data suggest that trauma severity and gender affect the reservoir and proliferation capacity of bone marrow-derived MSCs.     

Effect of Age and Ovariectomy on Fibroblastic Colony-Forming Unit Numbers in Rat Bone Marrow

SUMMARY. It is now thought that osteoblasts (OB) stem from mesenchymal precursor cells (fibroblastic colony forming units or CFU-f) in the bone marrow. As the availability of these cells may have a profound effect on bone formation, the effect of age and ovariectomy (OVX) on CFU-f numbers was studied. It was found that with increasing age the numbers of CFU-f in the marrow were drastically reduced. OVX had no effect on CFU-f levels in 4 week and 4 month old rats but raised CFU-f levels in 1 year old rats back to levels seen in younger rats. Sham operation alone had an effect on CFU-f levels in older rats and the OVX effects were indistinguishable from the sham effect until 20 d post operation when the sham levels returned to baseline. The results show that CFU-f levels follow a similar trend to the rates of bone formation seen in aged and OVX rats. Received: 7 July 1995 / Accepted: 28 May 1996     

Optimising proliferation and migration of mesenchymal stem cells using platelet products: A rational approach to bone regeneration

This study investigates how mesenchymal stem cell's (MSCs) proliferation and migration abilities are influenced by various platelet products (PP). Donor‐matched, clinical‐, and control laboratory‐standard PPs were generated and assessed based on their platelet and leukocyte concentrations. Bone marrow derived MSCs were exposed to these PP to quantify their effect on in vitro MSC proliferation and migration. An adapted colony forming unit fibroblast (CFU‐F) assay was carried out on bone marrow aspirate using clinical‐standard PP‐loaded electrospun poly(?‐caprolactone) (PCL) membrane to mimic future clinical applications to contain bone defects. Clinical‐standard PP had lower platelet (2.5 fold, p < 0.0001) and higher leukocyte (14.1 fold, p < 0.0001) concentrations compared to laboratory‐standard PP. It induced suboptimal MSC proliferation compared to laboratory‐standard PP and fetal calf serum (FCS). All PP induced significantly more MSC migration than FCS up to 24 h. The removal of leukocytes from PP had no effect on MSC proliferation or migration. The PP‐loaded membranes successfully supported MSC colony formation. This study indicates that platelet concentrations in PP impact MSC proliferation more than the presence of leukocytes, whilst MSC migration in response to PP is not influenced by platelet or leukocyte numbers. Clinical‐standard PP could be applied alongside manufactured membranes in the future treatment of bone reconstruction. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1329–1338, 2019.     

Disease modification of breast cancer–induced bone remodeling by cannabinoid 2 receptor agonists

Most commonly originating from breast malignancies, metastatic bone cancer causes bone destruction and severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain, and drug‐induced side effects; furthermore, recent findings suggest that patients are severely undertreated for their cancer pain. Strong analgesics, namely opiates, are first‐line therapy in alleviating cancer‐related pain despite the severe side effects, including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which are associated with highly undesirable side effects, including nephrotoxicity and osteonecrosis of the jaw. In contrast, cannabinoid receptor 2 (CB₂) receptor‐specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB₂ agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB₁/CB₂ agonists also demonstrate a reduction in ErbB2‐driven breast cancer progression. Here we demonstrate for the first time that CB₂ agonists reduce breast cancer–induced bone pain, bone loss, and breast cancer proliferation via cytokine/chemokine suppression. Studies used the spontaneously‐occurring murine mammary cell line (66.1) implanted into the femur intramedullary space; measurements of spontaneous pain, bone loss, and cancer proliferation were made. The systemic administration of a CB₂ agonist, JWH015, for 7 days significantly attenuated bone remodeling, assuaged spontaneous pain, and decreased primary tumor burden. CB₂‐mediated effects in vivo were reversed by concurrent treatment with a CB₂ antagonist/inverse agonist but not with a CB₁ antagonist/inverse agonist. In vitro, JWH015 reduced cancer cell proliferation and inflammatory mediators that have been shown to promote pain, bone loss, and proliferation. Taken together, these results suggest CB₂ agonists as a novel treatment for breast cancer–induced bone pain, in which disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone pain, and increased survival without the major side effects of current therapeutic options. © 2013 American Society for Bone and Mineral Research     

Basic Fibroblast Growth Factor in the Presence of Dexamethasone Stimulates Colony Formation, Expansion, and Osteoblastic Differentiation by Rat Bone Marrow Stromal Cells

Basic fibroblast growth factor (bFGF) is known to stimulate endosteal bone formation in vivo by a mechanism possibly mediated via osteoblast precursor cells present in the bone marrow. In high density cultures of primary bone marrow cells, and in the presence of glucocorticoids, bFGF stimulates the formation of a bone-like matrix; however, due to the dense nature of these cultures, the exact mechanism of action is unclear. In an adaptation of the fibroblastic colony formation unit assay, in which the bone marrow cells are grown in the presence of dexamethasone, β-glycerophosphate, and ascorbate, mineralized colonies are formed which stem from single mesenchymal precursor cells and grow in isolation from each other. Using this system we have been able to investigate the mechanism by which bFGF stimulates the formation of bone like tissue in vitro. We have shown that bFGF increases the formation of a calcified collagenous matrix in vitro by (1) increasing the total number of fibroblastic colonies formed, (2) increasing the proportion of differentiated colonies that synthesize collagen and calcify, and (3) stimulating the proliferation and collagen accumulation of the individual colonies. A maximal increase in total and differentiated colony numbers was seen after only 5 days exposure to bFGF, however, continued exposure to bFGF continued to increase the size and collagen content of the individual colonies. Bearing in mind the endosteal location of newly formed bone seen after treatment with bFGF, these processes may well play an active role in this effect. Received: 17 January 1997 / Accepted: 30 July 1998     

Low‐dose BMP‐2 and MSC dual delivery onto coral scaffold for critical‐size bone defect regeneration in sheep

Tissue‐engineered constructs (TECs) combining resorbable calcium‐based scaffolds and mesenchymal stem cells (MSCs) have the capability to regenerate large bone defects. Inconsistent results have, however, been observed, with a lack of osteoinductivity as a possible cause of failure. This study aimed to evaluate the impact of the addition of low‐dose bone morphogenetic protein‐2 (BMP‐2) to MSC‐coral‐TECs on the healing of clinically relevant segmental bone defects in sheep. Coral granules were either seeded with autologous MSCs (bone marrow‐derived) or loaded with BMP‐2. A 25‐mm‐long metatarsal bone defect was created and stabilized with a plate in 18 sheep. Defects were filled with one of the following TECs: (i) BMP (n = 5); (ii) MSC (n = 7); or (iii) MSC‐BMP (n = 6). Radiographic follow‐up was performed until animal sacrifice at 4 months. Bone formation and scaffold resorption were assessed by micro‐CT and histological analysis. Bone union with nearly complete scaffold resorption was observed in 1/5, 2/7, and 3/6 animals, when BMP‐, MSC‐, and MSC‐BMP‐TECs were implanted, respectively. The amount of newly formed bone was not statistically different between groups: 1074 mm³ [970–2478 mm³], 1155 mm³ [970–2595 mm³], and 2343 mm³ [931–3276 mm³] for BMP‐, MSC‐, and MSC‐BMP‐TECs, respectively. Increased scaffold resorption rate using BMP‐TECs was the only potential side effect observed. In conclusion, although the dual delivery of MSCs and BMP‐2 onto a coral scaffold further increased bone formation and bone union when compared to single treatment, results were non‐significant. Only 50% of the defects healed, demonstrating the need for further refinement of this strategy before clinical use. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2637–2645, 2017.

     

Defects in mesenchymal stem cell self‐renewal and cell fate determination lead to an osteopenic phenotype in Bmi‐1 null mice

In parathyroid hormone–related protein 1‐84 [PTHrP(1‐84)] knockin mice, expression of the polycomb protein Bmi‐1 is reduced and potentially can mediate the phenotypic alterations observed. We have therefore now examined the skeletal phenotype of Bmi‐1^?/? mice in vivo and also assessed the function of bone marrow mesenchymal stem cells (BM‐MSCs) from Bmi‐1^?/? mice ex vivo in culture. Neonatal Bmi‐1^?/? mice exhibited skeletal growth retardation, with reduced chondrocyte proliferation and increased apoptosis. Osteoblast numbers; gene expression of alkaline phosphatase, type I collagen, and osteocalcin; the mineral apposition rate; trabecular bone volume; and bone mineral density all were reduced significantly; however, the number of bone marrow adipocytes and Ppar‐γ expression were increased. These changes were consistent with the skeletal phenotype observed in the PTHrP(1‐84) knockin mouse. The efficiency of colony‐forming unit fibroblast (CFU‐F) formation in bone marrow cultures was decreased, and the percentage of alkaline phosphatase–positive CFU‐F and Runx2 expression were reduced. In contrast, adipocyte formation and Ppar‐γ expression in cultures were increased, and expression of the polycomb protein sirtuin (Sirt1) was reduced. Reduced proliferation and increased apoptosis of BM‐MSCs were associated with upregulation of senescence‐associated tumor‐suppressor genes, including p16, p19, and p27. Analysis of the skeletal phenotype in Bmi‐1^?/? mice suggests that Bmi‐1 functions downstream of PTHrP. Furthermore, our studies indicate that Bmi‐1 maintains self‐renewal of BM‐MSCs by inhibiting the expression of p27, p16, and p19 and alters the cell fate of BM‐MSCs by enhancing osteoblast differentiation and inhibiting adipocyte differentiation at least in part by stimulating Sirt1 expression. Bmi‐1 therefore plays a critical role in promoting osteogenesis. © 2010 American Society for Bone and Mineral Research     

Use of Allogeneic Hypoxic Mesenchymal Stem Cells For Treating Disc Degeneration in Rabbits

Intervertebral discs (IVDs) are important biomechanical components of the spine. Once degenerated, mesenchymal stem cell (MSC)‐based therapies may aid in the repair of these discs. Although hypoxic preconditioning enhances the chondrogenic potential of MSCs, it is unknown whether bone marrow MSCs expanded under hypoxic conditions (1% O₂, here referred to as hypoxic MSCs) are better than bone marrow MSCs expanded under normoxic conditions (air, here referred to as normoxic MSCs) with regards to disc regeneration capacity. The purpose of this study was to compare the therapeutic effects of hypoxic and normoxic MSCs in a rabbit needle puncture degenerated disc model after intra‐disc injection. Six weeks after needle puncture, MSCs were injected into the IVD. A vehicle‐treated group and an un‐punctured sham‐control group were included as controls. The tissues were analyzed by histological and immunohistochemical methods 6 and 12 weeks post‐injection. At 6 and 12 weeks, less disc space narrowing was evident in the hypoxic MSC‐treated group compared to the normoxic MSC‐treated group. Significantly better histological scores were observed in the hypoxic MSC group. Discs treated with hypoxic MSCs also demonstrated significantly better extracellular matrix deposition in type II and XI collagen. Increased CD105 and BMP‐7 expression were also observed upon injection of hypoxic MSCs. In conclusion, hypoxic MSC injection was more effective than normoxic MSC injection for reducing IVD degeneration progression in vivo. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1440–1450, 2019.     

Adenosine receptor subtype expression and activation influence the differentiation of mesenchymal stem cells to osteoblasts and adipocytes

Osteoblasts and adipocytes differentiate from a common precursor cell, the mesenchymal stem cell (MSC). Adenosine is known to signal via four adenosine receptor subtypes, and significantly, recent findings indicate that these may play a role in MSC differentiation. We therefore investigated adenosine receptor expression and activation during the differentiation of MSCs to osteoblasts and adipocytes. The A_2BR was dominant in MSCs, and its expression and activity were transiently upregulated at early stages of osteoblastic differentiation. Both activation and overexpression of A_2BR induced the expression of osteoblast‐related genes [Runx2 and alkaline phosphatase (ALP)], as well as ALP activity, and stimulation increased osteoblast mineralization. The expression of A_2AR was upregulated during later stages of osteoblastic differentiation, when its activation stimulated ALP activity. Differentiation of MSCs to adipocytes was accompanied by significant increases in A₁R and A_2AR expression, and their activation was associated with increased adipogenesis. Enhanced A_2AR expression was sufficient to promote expression of adipocyte‐related genes (PPARγ and C/EBPα), and its activation resulted in increased adipocytic differentiation and lipid accumulation. In contrast, the A₁R was involved mainly in lipogenic activity of adipocytes rather than in their differentiation. These results show that adenosine receptors are differentially expressed and involved in lineage‐specific differentiation of MSCs. We conclude, therefore, that fruitful strategies for treating diseases associated with an imbalance in the differentiation and function of these lineages should include targeting adenosine receptor signal pathways. Specifically, these research avenues will be useful in preventing or treating conditions with insufficient bone or excessive adipocyte formation. © 2011 American Society for Bone and Mineral Research     

Development and characterization of novel clinical grade neonatal porcine bone marrow‐derived mesenchymal stem cells

Due to recent advances in research on mesenchymal stem cells (MSCs), MSCs are expected to be used in various clinical applications. However, securing adequate cadaveric donors and safety of living donors are major issues. To solve such issues, we have examined to develop clinical grade neonatal porcine bone marrow‐derived MSCs (npBM‐MSCs). Clinical grade neonatal porcine bone marrow cells were collected, frozen, and sent to our laboratory by air. The npBM‐MSCs were isolated from thawed bone marrow cells, then frozen. The thawed npBM‐MSCs were examined for CD markers and differentiated into chondrocytes, osteocytes, and adipocytes. They were compared with human bone marrow‐derived MSCs (hBM‐MSCs) for growth rate and size. To assess the robustness of proliferation, we compared culture medium with or without gelatin. The npBM‐MSCs expressed positive MSC markers CD29, CD44, and CD90 and were differentiated into chondrocytes, osteocytes, and adipocytes. The doubling time of npBM‐MSCs was significantly shorter than that of hBM‐MSCs (17.3 ± 0.8 vs 62.0 ± 19.6 hours, P < 0.01). The size of npBM‐MSCs was also significantly smaller than that of hBM‐MSCs (13.1 ± 0.3 vs 17.5 ± 0.4 μm, P < 0.001). The npBM‐MSCs showed similar proliferation characters irrespective of with or without gelatin coating. The npBM‐MSCs secreted VEGF‐A, VEGF‐C, and TGF‐β1. We have established npBM‐MSCs which show super‐rapid growth, small size, and robust proliferation profile. The np‐MSCs might be able to solve the donor issues for MSC therapy.     

Native multipotential stromal cell colonization and graft expander potential of a bovine natural bone scaffold

Graft expanders are bone scaffolds used, in combination with autografts, to fill large bone defects in trauma surgery. This study investigates the graft expander potential of a natural bone substitute Orthoss® by studying its ability to support attachment, growth and osteogenic differentiation of neighboring multipotential stromal cells (MSCs). Material consisting of bone marrow (BM) aspirate and reamer‐irrigator‐aspirator (RIA)‐harvested autograft bone was co‐cultured with commercially available Orthoss® granules. Native MSCs attached to Orthoss® were expanded and phenotypically characterized. MSCs egress from neighboring cancelous bone was assessed in 3D Matrigel co‐cultures. MSC differentiation was evaluated using scanning electron microscopy and measuring alkaline phosphatase (ALP) activity per cell. CD45⁺ hematopoietic lineage cells and highly proliferative CD90⁺CD73⁺CD105⁺ MSCs preferentially colonized Orthoss® granules, over RIA bone chips. MSC colonization was followed by their intrinsic osteogenic differentiation, assessed as mineral deposition and gradual rise in ALP activity, even in the absence of osteogenic stimuli. When in contact with mixed cell populations and RIA chips, Orthoss® granules support the attachment, growth and osteogenic differentiation of neighboring MSCs. Therefore, natural bone substitutes similar to Orthoss® can be used as void fillers and graft expanders for repairing large bone defects in conjunction with autologous BM aspirates and autografts. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1950–1958, 2013     

Comparison of the osteogenic capacity of minipig and human bone marrow‐derived mesenchymal stem cells

Minipigs are a recommended large animal model for preclinical testing of human orthopedic implants. Mesenchymal stem cells (MSCs) are the key repair cells in bone healing and implant osseointegration, but the osteogenic capacity of minipig MSCs is incompletely known. The aim of this study was to isolate and characterize minipig bone marrow (BM) and peripheral blood (PB) MSCs in comparison to human BM‐MSCs. BM sample was aspirated from posterior iliac crest of five male Göttingen minipigs (age 15 ± 1 months). PB sample was drawn for isolation of circulating MSCs. MSCs were selected by plastic‐adherence as originally described by Friedenstein. Cell morphology, colony formation, proliferation, surface marker expression, and differentiation were examined. Human BM‐MSCs were isolated and cultured from adult fracture patients (n = 13, age 19–60 years) using identical techniques. MSCs were found in all minipig BM samples, but no circulating MSCs could be detected. Minipig BM‐MSCs had similar morphology, proliferation, and colony formation capacities as human BM‐MSCs. Unexpectedly, minipig BM‐MSCs had a significantly lower ability than human BM‐MSCs to form differentiated and functional osteoblasts. This observation emphasizes the need for species‐specific optimization of MSC culture protocol before direct systematic comparison of MSCs between human and various preclinical large animal models can be made. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1019–1025, 2012     

Effect of vitamin K<Subscript>2</Subscript> and growth hormone on the long bones in hypophysectomized young rats: a bone histomorphometry study

The purpose of the present study was to determine whether vitamin K₂ and growth hormone (GH) had an additive effect on the long bones in hypophysectomized young rats. Forty-eight female Sprague–Dawley rats (6 weeks old) were assigned to the following five groups by the stratified weight randomization method: intact controls, hypophysectomy (HX) alone, HX + vitamin K₂ (30 mg/kg, p.o., daily), HX + GH (0.625 mg/kg, s.c., 5 days a week), and HX + vitamin K₂ + GH. The duration of the experiment was 4 weeks. HX resulted in a reduction of the cancellous bone volume/total tissue volume (BV/TV) at the proximal tibial metaphysis, as well as decreasing the total tissue area and cortical area of the tibial diaphysis. These changes resulted from a decrease of the longitudinal growth rate and the bone formation rate (BFR)/TV of cancellous bone, as well as a decrease of the periosteal BFR/bone surface (BS) and an increase of endocortical bone turnover (indicated by the BFR/BS) in cortical bone. Administration of vitamin K₂ to HX rats did not affect the cancellous BV/TV or the cortical area. On the other hand, GH completely prevented the decrease of total tissue area and cortical area in cortical bone, as well as the decrease of marrow area and endocortical circumference, by increasing the periosteal BFR/BS compared with that in intact controls and reversing the increase of endocortical bone turnover (BFR/BS). However, GH only partly improved the reduction of the cancellous BV/TV, despite an increase of the longitudinal growth rate and BFR/TV compared with those of intact controls. When administered with GH, vitamin K₂ counteracted the reduction of endocortical bone turnover (BFR/BS) and circumference caused by GH treatment, resulting in no significant difference of marrow area from that in untreated HX rats. These results suggest that, despite the lack of an obvious effect on bone parameters, vitamin K₂ normalizes the size of the marrow cavity during development of the bone marrow in young HX rats treated with GH.