Subjects included in the present study are all patients who underwent an MSC-based, percutaneous injection treatment of an orthopaedic condition between December 2005 and September 2014 at one of 18 clinical facilities located in the United States or Australia and who had attained at least a three month follow-up period. Treated conditions included those resulting from degenerative joint changes (i.e., osteoarthritis, degenerative disc disease, degenerative disc disease) as well as trauma (e.g., anterior cruciate ligament injuries, rotator cuff tears, etc.). Treated areas of the body included the knee, hip, ankle/foot, hand/wrist, elbow, shoulder, and spine. Knee, hip, and shoulder patients constituted approximately 87 % of the population.

The patients were followed prospectively via enrollment in a treatment registry. Patients were grouped by type of MSC treatment (see below). The choice of treatment type was left to the treating physician and while there were no exclusion criteria for MSC-treated patients to enter the registry, patients were naturally excluded from treatment if they were found not to be a candidate for the treatment by the attending physician. Reasons for exclusion from treatment included conditions for which the only therapeutic alternative was deemed to be surgery as well as medical conditions that would make MSC therapy difficult. Examples include a completely torn and retracted tendon or ligament, a severely osteoarthritic knee with deformity, severe spinal stenosis with neurologic compromise, and severe rheumatologic conditions like rheumatoid arthritis or systemic lupus erythematosus. Institutional Review Board oversight for the registry protocol was provided by a U.S. Office of Human Research Protections registered organization (#IRB00002637). Outcomes and efficacy of each procedure have been reported previously [2, 8, 13, 16]. Prior to each procedure, physicians discussed risks, benefits, and alternatives to the procedure. Each subject gave both oral and written informed consent for procedure.

Baseline information collected in the registry included primary diagnosis, patient demographics, medical history, and physical examination. Patients were followed prospectively at 1, 3, 6, and 12 months post treatment, and annually thereafter, using an electronic system, ClinCapture software (Clinovo Clinical Data Solutions, Sunnyvale, California). Patients were sent automated e-mails that asked them to respond to a number of questions regarding outcomes, function, and general health. Three e-mails were sent once a week and if the patient failed to respond after three e-mails, the registry staff initiated two phone calls. If the patient failed to respond to these additional two queries, then the time point was considered lost to follow-up and the process began again at the next time point. Attending physicians participating in the registry were also encouraged to report any complications.

In addition to outcome information, patients were also asked the following two questions regarding possible treatment-related adverse events (AEs): “Did you experience any complications you believe may be due to the procedure (i.e., infection, illness, etc.)? If yes, please explain;” and “Have you been diagnosed with any new illness since the procedure? If yes, please explain.” The complications questions were intentionally broad in order to capture any change in the patient’s health status that could possibly be related to the MSC procedure.

The patients were grouped based on type of MSC treatment, as follows: SD (same day aspiration, isolation, and re-injection procedure with BMC), AD (same day aspiration, isolation, and re-injection procedure with BMC plus adipose graft), and CE (culture expanded MSCs re-implanted weeks or months after bone marrow aspiration) (see Supplement 1). All physicians were trained to use the same protocol for bone marrow aspiration, adipose graft, and re-injection procedures.

Two weeks prior to the bone marrow harvest procedure, patients in all groups were restricted from using steroidal and non-steroidal anti-inflammatory drugs in order to avoid possible cytotoxic effects on MSCs [17]. All injections in this study were confirmed with ultrasound or fluoroscopic imaging to ensure accurate placement. Two to five days prior to the administration of the MSCs to the treatment area, the patient’s joint, ligament, or tendon was pre-injected with 12.5 % hypertonic dextrose to promote an inflammatory response and begin the process of tissue repair. The decision to use this protocol was based on promising earlier observations in animal models that this protocol aided tendon healing and improved function in knee osteoarthritis patients and confirmed more recently through stabilization of cartilage volume on MRI in patients receiving only this treatment [18]. A detailed description of the procedures performed for the SD, AD, and CE groups are provided in our earlier publications [7, 9, 16]. Briefly, bone marrow harvest was completed via the collection of approximately 10–15 cc of bone marrow aspirate from the six to ten total sites from the bilateral posterior iliac crests. For the BMC injections (SD and AD groups), the aspirate was centrifuged to separate the buffy coat, resulting in 1–3 ml of BMC generally containing 0.2-1.5 × 10⁸ nucleated cells. Platelet rich plasma (PRP) and platelet lysate (PL) was concurrently prepared and injected along with the BMC into the target region on the same day as the bone marrow aspiration. In the AD group, an additional component of minimally processed lipo-aspirate which had been separated from the aqueous and oil components was co-injected along with the BMC (3–7 cc) and PRP and PL solution [7]. All isolation techniques for PRP, PL, SD, AD, and CE were standardized using a standard operating procedure (SOP) protocol that has been described in previous publications [7, 9, 16]. Specifically, purpose built kits were not used, but all sites used the same off the shelf disposable lab supplies and the same or similar equipment such as centrifuges, pipettes, and microscopy. Staff at each site were trained in these SOP protocols. Based off the PLRA classification, the type of PRP produced is 1 cc of 14x/−/−/NO [19] but baseline platelet counts were not obtained. In the CE group, MSCs isolated from the bone marrow aspirate were expanded in an autologous based culture media for 12–16 days prior to injection into the joint space (1–3 cc in PL with dose ranges generally from 0.1-6 × 10⁷ MSCs) or musculoskeletal structure (see Supplement 1 which elaborates on treatment differences between groups) [14]. Injectate volumes and dose were recorded, but not controlled and were determined by the treating physician.

AEs accessed from the treatment registry were initially sorted into one of 20 categories: allergic, bone, cardiac, endocrine, gastrointestinal, immune, infection, lab work, neoplasm, neurologic, pain-post procedure, pain due to progressive DJD, pain-other areas, pain-other, pulmonary, renal, rheumatological, skin, vascular, and other.

AEs were further categorized by the attending physician as: (1) serious adverse events (SAEs) or non-SAEs (2) expected or unexpected, and, as appropriate (3) related to the implantation procedure or related to stem cells (not mutually exclusive). AEs related to the implantation procedure or the stem cells were further defined as “definite,” “possible,” “unlikely,” or “not related.” SAEs were defined using guidelines developed by the United States Department of Health and Human Services [20]. This is defined as any untoward event that results in death, is life-threatening, requires inpatient hospitalization or causes prolongation of existing hospitalization, results in persistent or significant disability/incapacity, or requires intervention to prevent permanent impairment or damage. All “possible” SAEs were tabulated by one author (CJC) and then provided to five independent physician reviewers who were blinded to any initial or subsequent adjudication by another reviewer. The tabulating author (CJC) also remained blinded as to the identity of the physician who performed any specific independent adjudication. The independent reviewers were unrelated to the treating physicians in the study. Independent reviewers were recruited via an electronic discussion board for physicians if they: (1) had experience in using platelet rich plasma or stem cells for orthopaedic conditions (2) were a practicing physician in private or academic practice (Mishra, Feb 2009). In order to estimate the AE incidence, a person-time metric was calculated based on the number of patients and the amount of time they were followed from the time of treatment. The follow-up period was calculated from the date of the procedure to the date of data access or study exit.

The treatment groups were described by age, body-mass index (BMI), follow-up time, gender, and the joint/area treated. Frequency, proportion, and the rate of AEs by category were reported for each treatment group. AE rates were compared between treatment groups using a chi-square test. Frequency, proportion, and rate were also reported for SAEs, expected AEs, procedure-related AEs, and stem cell-related AEs. Categorical differences in proportions and rates between groups were analyzed using a chi-square test. Post hoc pair-wise comparisons between groups were made using chi-square or Fisher’s exact test, as appropriate. AE incidence rates were calculated by dividing the frequency of a specific AE by the total person-year (PY) denominator, with the results reported per 100 PY. Logistic regression analysis for binary outcomes was used to quantify the risk of reporting an AE, SAE, and treatment-related AE by treatment group, and adjusted for potential predictive or confounding factors (i.e., length of follow-up, age, gender, and body area treated). All statistical analyses were performed using SAS software version 9.4 [21].