Correlation of Age and Bone Marrow Derived CD 34+ Cells and Leucocytes in 873 Patients.

Background: The use of regenerative medicine, such as autologous chondrocyte implantation (ACI), matrix associated stem cell therapy (MAST) and bone marrow derived stem cell therapy against arthritis is the gold standard for certain indications. However, the clinical improvement of patients using these novel therapies remains heterogeneous and the reasons for this are not fully understood. The impact of age is always a concern for patients and doctors and elderly patients can only be mobilized with lower total collected CD34+ cells, older age correlates with inferior results, fatty degeneration of the bone marrow, delayed fracture-healing and osteoporosis


Introduction
The use of cell therapy, such as autologous chondrocyte implantation (ACI) and matrix associated stem cell therapy (MAST) for the repair of damaged cartilage is well established, demonstrating good short to medium term outcomes. 9,24 owever, the clinical improvement of patients using these novel therapies remains heterogenous and the effects are not fully understood.Recently bone marrow derived stem cells gained further attraction in orthopedic diagnoses, where they are also used as bone marrow aspirate (BMA) or bone marrow aspirate concentration (BMAC) for stem cell therapy.
(1) Arthritis is treated in this way, aiming to generate additional cartilage, and stop the in ammatory process. 8,11,12,18,27,31,33,35 (2) Intients with spinal cord injuries, bone marrow derived stem cells can improve some neural function. 6,7,13,15,17,23 Regaess of the purpose of a cell therapy, successful cell transplantations requires the use of a su cient number of speci c cells and their engraftment. 20Better clinical results can be achieved with a higher donor-site stem cell count 19,30 and a lower age. 14The impact of age is not fully understood for stem cell procedures and controversial: Speci c qualitative age-dependent ndings were published up to now (lower proliferation and extra cellular matrix forming potential 16 , decreasing growth rate and telomere length, 4 lower mobilization rate, 1,28 fatty degeneration of the bone marrow, delayed fracture-healing and osteoporosis, 21 acquired mitochondrial DNA mutations 34 ) but the quantity of stem cells according to age has not yet been studied su ciently.In hemato-oncology is is known, that grafts from older donors do not adversely affect outcomes of allogeneic hematopoietic cell transplantation as compared to grafts from younger donors in. 10,28 ce an age-dependent range of the physiological leukocyte-and stem-cell-numbers and their vitality in the human bone marrow has not yet been reported and most clinical studies about CD 34+ stem cells refer to patients with malignant diseases, we wanted to establish a normal range of bone marrow derived leukocytes and stem cells of patients without malignancies according to age, harvested by bone amrrow aspiration (BMA).
Therefore the aim of this study is, to evaluate the number and the vitality of bone marrow derived leukocytes and CD 34+ cells in a large number of patients undergoing autologous stem cell transplantation for nonmalignant diseases study the connection to age in order to nd out, if a possible age limit is present, after which cell number and vitality decreases.
predictor exists for the amount of CD 34+ cells in the bone marrrow.

Level IIb: Retrospective cohort study
In a retrospective study the laboratory results all patients, who underwent stem stell transplantation for non malignant diseases were evaluated.All bone marrow punctures were done by the same surgeon (K.G.).The stem cells were harvested with a Yamshidi Needle (15 ga x 2.688in MAX Bone Marrow Aspiration Needle, ARGON Medical devices, Athens, USA, www.argonmedical.com)under sedoanalgesia and in compliance with all applicable laws and regulations.90ml of bone marrow aspirate was retrieved using the Technique of Kristin Oliver 26 (using 10ml syringes and changing direction repeatedly).One milliliter of this sample was immediately transferred to a laboratory and analyzed with FACS (Fluorescence Activated Cell sorter) using a stem Cell Kit from Beckman Coulter and the ISHAGE protocol (https://www.bc-cytometry.com/PDF/DataSheet/IM3630.pdf).Results 873 datasets were found in the laboratory patient database.Age ranged from 1-90 years (mean 28, median 25) and patients were clustered into age groups of 10 years.Gender distribution was 29% female and 71% male.There was no gender-difference.The anonymous laboratory data was statistically evaluated by a blinded observer.(Table 1) Table 1 Age distribution of bone marrow derived cells

Bone marrow derived leucocyte cell count
No signi cant differences between age and gender and no interaction.Even in a pairwise comparison of patients below the age of 20 versus patients at the age of 20 years and over, no signi cant differences were found (p=.9).The correlation between age and bone marrow derived leucocyte cell count is negligible (r=-.255,p<.001, Figure 1).

Bone marrow derived leucocyte vitality
No signi cant differences between age and gender and no interaction in all age groups, no signi cant correlation.(Table 1)

Bone marrow derived CD34+ cell count
No signi cant differences between age and gender and no interaction.Even in a pairwise comparison of patients below the age of 20 versus patients at the age of 20 years and over, no signi cant differences were found (p=.8).The correlation between age and bone marrow derived CD 34+ cell count is negligible and comes from patients below the age of 10 years (r=-.361,p<.001, Figure 2).

Correlation / Predictor
The number of bone marrow derived leucocytes and CD 34+ cells (as a subset of leucocytes, respectively) had a great variation between individual patients, but both cell types correlated strong and signi cant (p<.001, r 2 =822, Figure 3) within the respective patients.Bone marrow derived leukocytes are therefore a viable predictor for the amount of stem cells.The number of stem cells can be calculated as follows: Stem cells (CD 34+ cells / Microliter) = 13.5 x Leukocytes (per Nanoliter bone marrow aspirate).
Bone marrow derived leucocytes and and CD 34+ cells had a negligible tendency (r=-.255,p<.001 and r=-.361, p<.001) to decrease over life time.The weak correlation was only due to the group of children below the age of 10 years.Thereafter there was a variation between individual patients, but no decrease over time.

Discussion
Arthritis and musculoskeletal disorders constitute a major cause of disability and the burden of musculoskeletal diseases will increase with an increasing ageing population. 22Stem cells remain at the forefront of efforts in Regenerative Medicine, based on a conviction that this technology can provide an effective treatment paradigm for major diseases where there is still an unmet need. 3 2017 the rst prospective, single blind, placebo-controlled trial of bone marrow aspirate concentrate for knee osteoarthritis described a positive clinical outcome. 31A recent review of 1500 papers on stem cell therapy in orthopaedics revealed, that studies reported information on only 42% (range, 25%-60%) of the variables included within established minimum reporting standards, leaving it unclear, which amount of stem cells was really harvested and injected. 25,29  higher donor-site cell count correlates with a better outcome. 8,14,18,19,27,35 In hmato-oncology, where bone marrow transplantations are performed routinely since decades, elderly patients show inferior mobilization rates and inferior outcome in some studies. 1,28,14 ikewise, in a mice model an age-related fatty degeneration of the bone marrow was described. 21e current study looked at the donor site of 873 healthy patients (without bone marrow diseases), applicable for orthopaedic interventions, to understand the vitality and the quality of the bone marrow derived (stem) cells.

Age and cell counts
Due to its large size this study is the rst to establish a normal range of leukocytes and CD34+ stem cells in bone marrow aspirate (BMA) in the average population.The amount and vitality of bone marrow derived leucocytes and the number of mononuclear cells (stem cells) do not deteriorate over age.
The formerly described lower mobilization rate of bone marrow derived (stem) cells in the elderly in some studies 1,28 con icts with a recent study, where little of the parameter variability could be explained by age. 5 Our nding, that the number of bone marrow derived leucocytes remains stable in adults was con rmed in a prior study with 24 goats, but we did not nd further papers referring to humans. 2

Vitality
The vitality of bone marrow derived cells was always high (87-91%) in all age groups using FACS ( uorescence-activated cell sorting) analysis.Regarding dental pulp stem, the proliferation rate decreases in elderly patients. 36We could not con rm this nding in bone marrow derived cells.It is well known, that jawbones have an different bone metabolism. 32rrelation / Predictor Stem cells are mononuclear cells and therefore a 7.4% fraction (1 / 13.5) of the bone marrow derived leucocytes, as we demonstrated.
To our knowledge no prior study described the positive and strong correlation (p<.001, r 2 =822) between both parameters.Since stem cells can only be identi ed using speci c CD antigen sets (CD 34, CD 90, CD 45, CD 107,…), which is costly and laborious, this correlation can be utilized, to predict the amount of stem cells based on the number of bone marrow derived leucocytes alone, which is much easier.As a matter of fact, only a negligible share of publications reports the absolute number of stem cells used per patient. 29Using the new described correlation, a much cheaper possibility exists, to assess, if a speci c patient has a high or low stem cell number, since counting leukocytes can be done in any operating room, but counting stem cells requires at least a FACS analysis.

Limitations:
Since this was a retrospective study, we were not able to assess any social data (body weight, sport habits, smoking status,…).We did not perform colonization and differentiation experiments, since the lack of a speci c MSC marker and the low frequency of MSCs in bone marrow necessitate their isolation by in vitro expansion.
Further research is needed to link clinical outcome with the absolute number of bone marrow derived leukocytes and stem cells since it is not yet clear, if the heterogenous clinical results of individual patients are linked to the heterogenous bone marrow derived stem cell counts.It remains speculative if patients with higher cell counts will have better outcomes and might therefore be better suitable for stem cell operations.

Conclusion
We established a normal range of bone marrow derived leukocytes and stem cells in 873 patients.No age-related decrease regarding the number and the vitality of leukocytes and stem cells was found.Furthermore the number of bone marrow derived leucocytes might be used to predict the amount of stem cells (usually a 7.4% share) on an individual basis in order to focus on the "ideal" patients.

Declarations
Ethics approval and consent to participate Use of anonymous secondary data, no trace to individual patients is possible .According to the declaration of Helsinky and the Ethics Commission of our University no informed consent necessary.

Consent for publication
Approved by all authors.There is no relevant decrease of bone marrow derived leucocyte cell count with increasing age.A strong correlation between bone marrow derived leucocytes and bone marrow derived CD34+ cells was found.
Prior bone marrow stimulation or treatment with GCSF (granulocyte colony stimulating factor) Prior treatment with hormons or corticoids in the last 6 months Statistical Analysis was done using: Pearson's chi-squared tests One-way analyses of variance (with post-hoc Bonferroni-adjusted pairwise comparisons of estimated marginal means) Pearson`s Correlation

Figure 2 Even
Figure 2