Reversing bone loss by directing mesenchymal stem cells to bone

Abstract

Bone regeneration by systemic transplantation of mesenchymal stem cells (MSCs) is problematic due to the inability to control the MSCs' commitment, growth, and differentiation into functional osteoblasts on the bone surface. Our research group has developed a method to direct the MSCs to the bone surface by conjugating a synthetic peptidomimetic ligand (LLP2A) that has high affinity for activated α4β1 integrin on the MSC surface, with a bisphosphonates (alendronate) that has high affinity for bone (LLP2A-Ale), to direct the transplanted MSCs to bone. Our in vitro experiments demonstrated that mobilization of LLP2A-Ale to hydroxyapatite accelerated MSC migration that was associated with an increase in the phosphorylation of Akt kinase and osteoblastogenesis. LLP2A-Ale increased the homing of the transplanted MSCs to bone as well as the osteoblast surface, significantly increased the rate of bone formation and restored both trabecular and cortical bone loss induced by estrogen deficiency or advanced age in mice. These results support LLP2A-Ale as a novel therapeutic option to direct the transplanted MSCs to bone for the treatment of established bone loss related to hormone deficiency and aging.

Keywords: Bone marrow stromal cells; Cell migration; In vivo tracking; Integrins; Stem cell transplantation.

Figure 1

LLP2A-Ale induced MSC migration was matrix and mineral dependent. A, Mouse bone marrow-derived MSCs were seeded in the upper chamber of transwell plates with inserts that were pre-coated with bovine serum albumin (BSA) or 10µg/ml collagen type I (Col I), fibronectin (FN), laminin (LAM), or collagen type IV (Col IV) in serum-free conditions. The bottom wells were loaded with either media alone or containing 45 nM LLP2A-Ale. The cells that had migrated through the pores to the lower surface of the membrane were stained with crystal violet. Subsequently, stained cells were eluted from membranes and absorbance measure at 590nm. B, A similar set of experiments was performed as described in “A” except that the bottom wells were pre-coated with 10% hydroxyapatite. C, A similar set of experiments was performed as described in “A” that the upper chamber of transwell plates with inserts were pre-coated with 10 µg/ml Col I in serum-free conditions. The bottom wells were loaded with either media alone or containing 45 nM Alendronate (Ale), LLP2A or LLP2A-Ale. D, Same experiments were performed as described in “C” except that the bottom wells were pre-coated with 10% hydroxyapatite. E, Murine phosphorylated RTK arrays were used to examine Col I-induced RTK phosphorylation levels in MSC lysates without hydroxyapatite or with hydroxyapatite following LLP2A-Ale (45nM) for 2 hours. F, Western blotting analyses were performed for total Akt, rpS6, phosphorylated Akt, rpS6 and actin. G, Quantitation for F. H, MSCs were cultured with LLP2A-Ale for 10 days. Ratio of CFU-Ob/CFU-F was measured. I, Osteocalcin levels were measured in for media in experiment described in “H”. Experiments were performed in duplicate and for at least three times. Data are means ± SD. *: p<0.05 vs. control (BSA or PBS). All the studies were done in triplicate.

Figure 2

MSCs bone homing and osteoblast differentiation following LLP2A-Ale treatment. A, Representative decalcified vertebral body sections were obtained from ovariectomized mice that received three injections of MSC or MSC + LLP2A-Ale. The transplanted MSCs were accessed by anti-GFP staining (green) at week 14 of the study. B, Representative decalcified vertebral body sections were obtained from 24-week-old mice that received two injections of MSC or MSC + LLP2A-Ale. The transplanted MSCs were stained in green, osteocalcin were stained in red. Cells that were positive for both GFP and osteocalcin appeared in yellow. C, Representative decalcified vertebral body sections were obtained from 24-month-old mice that received two injections of MSC or MSC + LLP2A-Ale. The transplanted MSCs were stained in green, osteocalcin were stained in red. Cells that were positive for both GFP and osteocalcin appeared in yellow.

Figure 3

LLP2A-Ale or with the combination of MSC transplantation increased bone formation in OVX mice. Two-month-old C57BL/6 were ovariectomized (OVX) and left un-treated for four weeks. They were then treated with PBS, MSC (5 × 10⁵), LLP2A-Ale (0.9 nmol/mouse, IV at weeks 2, 6 and 10), MSC + LLP2A-Ale (LLP2A-Ale, 0.9 nmol/mouse; MSC 5 × 10⁵/mouse, IV at weeks 2, 6 and 10) or human PTH (–34) (30µg/kg, 3x/week). Mice were sacrificed at week 16. Alizarin red was injected prior to the treatments and two tetracycline injections were given 7 and 2 days before the mice were sacrificed. A, Osteoblast surface, bone formation rate, bone volume and maximum stress were measured from the lumbar vertebral bodies. B, Representative sections from the lumbar vertebral bodies that were stained in tetrachrome and von kossa or left unstained. Bone was stained in black. Green arrow heads illustrate osteoblasts at the bone surface. Blue arrow heads illustrates osteotoid bridge. Yellow arrows illustrate double tetracycline-labeled bone surfaced. C, Representative trabecular thickness maps were obtained from the distal femurs by micro-CT where the trabecular thickness is color coded with blue-green color-codes thinner trabeculae while yellow-red color-codes thicker trabeculae. D, Endocortical bone formation rate, cortical bone thickness, maximum load and ultimate strength were measured at the right femurs. Double-side white arrows illustrate total bone gain at the endocortical compartments during the treatment period. *: p<0.05 between the indicated groups.

Figure 4

LLP2A-Ale or with the combination of MSC transplantation increased trabecular and cortical bone formation and strength in adult mice. Twenty-four -week old female mice were treated with PBS, MSC (1× 10⁵), LLP2A-Ale (0.9 nmol/mouse, IV at baseline and week 6), MSC + LLP2A-Ale (LLP2A-Ale, 0.9 nmol/mouse; MSC 5 × 10⁵/mouse, IV at baseline and week 6) or human PTH (–34) (30µg/kg, 5x/week). Mice were sacrificed at week 12. Alizarin red was injected prior to the treatments and two calcein injections were given 9 and 2 days before the mice were sacrificed. A, Osteoblast surface, mineralizing surface, bone formation rate, bone volume, maximum load and stress were measured from the lumbar vertebral bodies. B, Representative sections that were stained in tetrachrome and von kossa or left unstained. Bone was stained in black. Green arrows illustrates osteoblasts at the bone surface, white arrows illustrate double calein-labeled bone surfaces. C, Representative cortical bone sections and D, bone formation rates measured at the endocortical and periosteal surfaces, ultimate strength of the femurs. White arrows illustrate double calein-labeled periosteal bone surface.*: p<0.05 between the indicated groups.

Figure 5

Combination treatment of MSC and LLP2A-Ale increased trabecular and cortical bone formation and bone volume in aged mice. Twenty-four -month old female mice were treated with PBS, MSC (5 × 10⁵), LLP2A-Ale (0.9 nmol/mouse, IV at baseline and week 6), MSC + LLP2A-Ale (LLP2A-Ale, 0.9 nmol/mouse; MSC 5 × 10⁵/mouse, IV at baseline and week six) or human PTH (–34) (30µg/kg, 5x/week). Mice were sacrificed at week 12. Alizarin red was injected prior to the treatments and two calcein injections were given 9 and 2 days before the mice were sacrificed. A, Osteoblast surface, mineralizing surface, bone formation rate, bone volume, maximum load and stress were measured from the lumbar vertebral bodies. B, Representative sections that were stained in tetrachrome and von kossa or left unstained. Bone was stained in black. Green arrows illustrates osteoblasts and osteoid bridging the trabeculae in the MSC + LLP2A-Ale treated group; white arrows illustrate double calein-labeled bone surfaces. C, Representative cortical bone sections and D, bone formation rates measured at the endocortical and periosteal surfaces, ultimate strength of the femurs. White arrows illustrate calein-labeled periosteal bone surface.*: p<0.05 between the indicated groups.