Mesenchymal stem cells (MSCs) are capable of forming bone, cartilage and other mesenchymal tissues but are also important modulators of innate and adaptive immune responses. We have capitalized on these important functions to mitigate adverse responses when bone is exposed to pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), or prolonged pro-inflammatory cytokines. Our goal was to optimize osteogenesis and mitigate persistent undesired inflammation by: 1. preconditioning MSCs by short term exposure to lipopolysaccharide (LPS) and Tumor Necrosis Factor alpha (TNF-α), 2. genetic modification of MSCs to overexpress Interleukin 4 (IL-4) either constitutively, or as NFκB-responsive IL-4 over-expression cells, and 3. training the MSCs (innate immune memory) by repeated stimulation with LPS.

In the first experiment, bone marrow MSCs and macrophages were isolated from femurs and tibias of C57BL/6 mice. MSCs (1×104 cells) were seeded in 24-well transwell plates in the bottom chamber with MSC growth medium. MSCs were treated with 20 ng/ml TNF-α and 1–20 μg/ml LPS for three days. Primary macrophages (2 × 103 cells) were seeded to the insert of a separate transwell plate and polarized into the M1 phenotype. At day four, MSCs and macrophages were washed and the inserts with M1 macrophages were moved to the plates containing preconditioned MSCs at the bottom of the well. Co-culture was carried out in MSC growth medium for 24h.

In the second experiment, bone marrow derived macrophages and MSCs were isolated from femora and tibiae of Balb/c male mice. 5×104 macrophages and 1×104 MSCs were seeded in the bottom well of the 24-well transwell plate. The upper chambers were seeded with unmodified MSCs, MSCs preconditioned with 20 ng/ml TNF-α and 20 mg/ml LPS for 3 days, NFκB-IL4 secreting MSCs (all 5×104 cells), or controls without MSCs. Co-culture was carried out in mixed osteogenic-macrophage media with clinically relevant polyethylene or titanium alloy particles.

In the third experiment, bone marrow MSCs and macrophages were collected from femurs and tibias of C57BL/6 male mice. The MSCs were stimulated by LPS, washed out for five days, and re-stimulated by LPS in co-culture with macrophages.

First, preconditioned MSCs enhanced anti-inflammatory M2 macrophage (Arginase 1 and CD206) expression, decreased pro-inflammatory M1 macrophage (TNF-α/IL-1Ra ratio) expression, and increased osteogenic markers (alkaline phosphatase expression and matrix mineralization) in co-culture. Second, NFκB-IL4 secreting MSCs decreased pro-inflammatory M1 (TNF-α), increased anti-inflammatory M2 (Arg1, IL-1ra) expression, and enhanced the expression of osteogenic factors Runx2 and alkaline phosphatase, in the presence of particles, compared to other groups.

Third, LPS-trained MSCs increased anti-inflammatory (Arginase1 and CD206), and decreased the proinflammatory (TNF-α, IL1b, iNOS, and IL6) marker expression in MSC/macrophage co-culture.

Transforming MSCs via the techniques of preconditioning, genetic modification, or training (innate immune memory) can modulate/convert a potentially injurious microenvironment to an anti-inflammatory pro-reconstructive milieu. These effects are highly relevant for bone healing in the presence of adverse stimuli. These concepts using transformed MSCs could also be extended to other organ systems subjected to potentially damaging agents.

Patients may present with concurrent symptomatic hip and spine problems, with surgical treatment indicated for both. Controversy exists over which procedure, total hip arthroplasty (THA) or lumbar spine procedure, should be performed first.

Clinical scenarios were devised for 5 fictional patients with both symptomatic hip and lumbar spine disorders for which surgical treatment was indicated. An email with survey link was sent to 110 clinical members of the NA Hip Society requesting responses to: which procedure should be performed first; the rationale for the decision with comments, and the type of THA prosthesis if “THA first” was chosen. The clinical scenarios were painful hip osteoarthritis and (1) lumbar spinal stenosis with neurologic claudication; (2) lumbar degenerative spondylolisthesis with leg pain; (3) lumbar disc herniation with leg weakness; (4) lumbar scoliosis with back pain; and (5) thoracolumbar disc herniation with myelopathy. Surgeon choices were compared among scenarios using chi-square analysis and comments analyzed using text mining.

Complete responses were received from 51 members (46%), with a mean of 30.8 (± 10.4) years of practice experience. The percentages of surgeons recommending “THA first” were 59% for scenario 1; 73% for scenario 2; 47% for scenario 3; 47% for scenario 4; and 10% for scenario 5 (χ²=44.5, p<0.001). Surgeons were significantly more likely to choose “THA first” despite radicular leg pain (scenario 2), and less likely to choose “THA first” with the presence of myelopathy (scenario 5). The choice of “THA first” in scenarios 1, 3, and 4 were more equivocal, dependent on surgeon impression of clinical severity. For type of THA prosthesis, dual mobility component was chosen by: 12% in scenario 1; 16% in scenario 2; 8% in scenario 3; 24% in scenario 4; and 10% in scenario 5. Surgeons were more likely to choose dual mobility in scenario 4, but with the numbers available this was not statistically significant (χ²=6.6, p=0.16). The analysis of comments suggested the importance of injection of the joint for decision making, the merit of predictable outcome with THA first, the concern of THA position with spinal deformity, and the urgency of myelopathy.

With the presence of concurrent hip and spine problems, the question of “THA or lumbar surgery first” remains controversial even for a group of experienced hip surgeons. Outcome studies of these patients are necessary for appropriate decision making.

Summary

Wear particles from joint replacements may result in loosening and periprosthetic osteolysis. Interference with systemic macrophage trafficking to the implant, modulation of macrophage phenotype from M1 to M2, and inhibition of NFκB may mitigate these adverse effects.