Introduction: Aging processes are connected to chronic inflammation, known as “inflammaging”. Age-associated inflammation is a critical predictor of age-related morbidity and mortality in older adults, specifically serum Tumor Necrosis Factor alpha (TNF-alpha) and interleukin (IL-6) levels. Mesenchymal Stem Cells (MSCs) and their exosomes can alleviate systemic inflammatory processes. We present a simple technique for harvesting autologous, peripheral MSC from venous blood together with their exosomes and we present a strategy of an intramuscular stem cell deposit combined with a three-dimensional Hyaluronic Acid (HA) matrix as scaffold for delayed stem cell release as an anti-inflammatory treatment for individuals with chronic systemic inflammatory conditions as possible strategy against inflammaging.

Material and Methods: Thirty milliliters (ml) venous whole blood are drawn from the cubital vein. By using a special centrifuge, 5 mL of peripheral MSC are harvested out of the venous whole blood together with 7 mL of MSC-derived exosomes and other anti-inflammatory autologous components. Then, we mix it with 88mg cross-linked hyaluronic acid (Monovisc, Anika Therapeutics, United States) that acts ad three-dimensional scaffold and as habitat for the MSCs and should lead to slowed release of the MSCs and their exosomes. After four weeks after injection of the 3D-stem cell deposit, serum levels of Interleukin-6 (IL-6), Tumor Necrosis Factor-Alpha (TNF-alpha), C-reactive Protein (CRP) were analyzed in 18 individuals with history of chronic systemic inflammatory diseases.

Results: We report on 35 individuals receiving the 3D-stem cell deposit between October 2023 and September 2024. There were no serious adverse effects or complications related to the injection of the 3D-stem cell deposit. All subjects receiving the 3D-stem cell deposit reported improvement of their general well-being within one week after the injection. Especially painful circumstances related to osteoarthritis, headache (migraine), lower back pain, cervical pain related to disc herniation, but also other inflammation-caused circumstances (atopic dermatitis, psoriasis, thyroid eye disease) improved subjectively as reported by all the individuals. All inflammation serum parameters significantly improved (dropped) after 4 weeks in all of the eighteen individuals analyzed.

Discussion: According to the results of our study and according to the fact that such pro-inflammatory parameters significantly decreased after administration of the 3D-stem cell deposit one can extrapolate the results suggesting lower mortality and extended lifespan after 3D-stem cell deposit administration. As a consequence and according to our data, the 3D-stem cell deposit might be an easy, effective and safe longevity concept by immunomodulation and by alleviating chronic inflammation.

Weninger P, Pfafl-Weninger V (2025) Intra-muscular, Ultrasound-guided Injection of Autologous, Peripheral Blood Stem Cells and Their Exosomes Combined with a Hyaluronic Acid Matrix Reduces Inflammaging – the “3d-Stem Cell Deposit” as a New Longevity Concept. J Aging Age Relat Dis 4(1): 1005.

Senescene and aging are inevitable and non-stoppable complex processes that lead to age-related diseases followed by cell death (apoptosis) and followed by the death of the individual. All biologic individuals are affected by the aging process and exposed to aging-related disorders such as skin wrinkles and sagging, hair loss, pigmental loss, cataract, cartilage degeneration and other organ dysfunctions as well as tumor growth and inflammatory processes [1-4]. Most of these circumstances are caused by loss of hydration, loss of collagen and other connective tissues, autophagy disorders, oxidative stress and mitochondrial dysfunction [5-8]. It is generally accepted that aging processes are connected to chronic inflammation, known as “inflammaging” [9-12]. Age-associated inflammation is a critical predictor of age-related morbidity and mortality in older adults, specifically serum tumor necrosis factor alpha (TNF-alpha) and interleukin (IL-6) levels [13,14].

By shifting the cytokine profile from anti-inflammation to inflammation, chronic inflammation has impact on development of age-related diseases and organ failure [15,16]. It is common knowledge that the amount of stem cells is decreasing with age and the amount of senescing cells increases [17,18]. The amount and also the renewal of Mesenchymal Stem Cells (MSC) are limited in the human body and loss of MSC can be seen as ultimate cause of the individual’s aging process [19,20]. Increasing the amount of viable MSCs within the individual’s body is known to postpone age-related processes by renewing organic structure and body’s tissue regeneration processes on cellular basis [21,22]. Numerous studies showed the role of MSCs in tissue regeneration, anti- aging and anti-inflammation, especially fast-turnover tissues such as the skin, bowel or haemato-poetic system [23-25]. Other target tissues are tissues primed on degeneration or necrosis after limited blood supply such as cartilage or cardiac tissue after myocardial infarction [26,27]. Furthermore, it was reported that systemic MSC administration has potential to alleviate autoimmune and inflammation-based systemic diseases [28,29]. It is generally accepted that MSCs have limited dwell time especially when administered intravenously [30-33]. It has been shown that MSC dwell time can be increased up to five months after intramuscular injection [34]. Based upon this fact we here present a simple technique for harvesting autologous, peripheral stem cells from venous blood together with their exosomes and we present a strategy of an intramuscular stem cell deposit combined with a three-dimensional Hyaluronic Acid (HA) matrix as scaffold for delayed MSC release as an anti-inflammatory treatment for individuals with chronic systemic inflammatory conditions as possible strategy against inflammaging.

This investigation was not designed as a clinical trial or study in advance and was defined as observational survey (“Anwendungsbeobachtung”). All individuals received treatment after informed consent was obtained as part of the treatment protocol for an individual therapy approach (“Individueller Heilversuch”) as contract between our institution and the individual subject. The use of autologous blood-derived products is generally legitimated by the federal agency AGES (Austrian Agency for Health and Food Safety) in form of a written statement as well as based on the document of the BASG (Bundesamt für Sicherheit im Gesundheitswesen, Dokument-Nr.: L_INS_VIE_ BTVI_I262_01) under “Single Use” procedures.

The patient is placed in supine position. Thirty milliliters (ml) venous whole blood are drawn from the cubital vein. The whole blood sample is transferred to a closed system cup by using a 17-gauge cannula (Figure 1).

Figure 1: 30mL whole blood after drawn from the cubital vein is inserted into the closed system cup before centrifugation.

Then, the closed system cup is placed in a centrifuge (SMART Cell, Miracell Ltd., South Korea) and is centrifuged at 2,500 for 17 minutes.

By using this special centrifuge, 5 mL of peripheral MSC are harvested out of the venous whole blood together with 7 mL of MSC-derived exosomes and other anti-inflammatory autologous components (Figure 2, Table 1).

Figure 2: MSCs (small red fraction) and MSC-derived exosomes (yellow fraction) after centrifugation. The erythrocytes (large red fraction) will be dismissed after centrifugation.

Table 1: Specification of peripheral blood stem cells and their exosomes.

Then, we mix it with 88mg cross- linked hyaluronic acid (Monovisc, Anika Therapeutics, United States) that acts ad three-dimensional scaffold and as habitat for the MSCs and should lead to slowed release of the MSCs and their exosomes. The skin, the subcutaneous layer and the rectus femoris muscle are anesthetized locally using 10 mL Lidocaine 2% with Epinephrine 1:10,000. Then, the 3D-stem cell deposit is injected into the rectus femoris muscle approximately 10 cm above the patella basis. The entire procedure is performed under ultrasound guidance by using a portable handheld ultrasound scanner (Clarius, Canada). Ultrasound is also used to monitor the size of the 3D-stem cell deposit every month to allow for decision making about the renewal of the deposit. In our practice and after administration of the 3D-stem cell deposit in 35 individuals the deposit was completely resorbed after six months in all individuals suggesting a repeated injection twice a year. After four weeks after injection of the 3D-stem cell deposit, serum levels of Interleukin-6 (IL-6), Tumor Necrosis Factor-Alpha (TNF-alpha), C-reactive Protein (CRP) were analyzed in 18 individuals with history of chronic systemic inflammatory diseases.

We report on 35 individuals receiving the 3D-stem cell deposit between October 2023 and September 2024. Demographic data and type of systemic inflammation is shown in table 2.

Table 2: Demographic data and causes for systemic inflammation.

There were no serious adverse effects or complications related to the injection of the 3D-stem cell deposit. In 4 individuals, mild local pain at the injection site was monitored after injection. No superficial or deep infections were detected. 3 patients developed mild hematoma at the puncture site. All subjects receiving the 3D-stem cell deposit reported improvement of their general well-being within one week after the injection. Especially painful circumstances related to osteoarthritis, headache (migraine), lower back pain, cervical pain related to disc herniation, but also other inflammation-caused circumstances (atopic dermatitis, psoriasis, thyroid eye disease) improved subjectively as reported by all the individuals. As demonstrated in figures 3-5,

Figure 3: IL-6 values before and after administration of the 3D-stemcell deposit.

Figure 4: TNF-alpha values before and after administration of the 3D-stemcell deposit.

Figure 5: CRP values before and after administration of the 3D-stemcell deposit.

all inflammation serum parameters significantly improved (dropped) after 4 weeks in all of the eighteen individuals analyzed.

Systemic administration of MSC and their exosomes are a reasonable option for tissue regeneration and anti-aging and also extends lifespan in mice [35-37]. To date, numerous studies are available, reporting the beneficial effects of MSC in regenerative medicine and reversed ageing. In a recent review, Zhang et al. highlighted the pathways and factors of aging as possible targets for MSC in anti-aging [38].

The importance of MSC as anti-aging strategy has also been pointed out by Weng et al. who emphasize the importance of cell viability and cell longevity [39].

In this context we also refer to the report of Braid et al. who as well described prolonged dwell time of MSC after intramuscular administration in a mouse model [34]. They showed longer cell survival after intramuscular injection compared to any other administration route for neonatal and adult MSC. In fact, they reported survival up to five months after intramuscular injection. This is also in accordance to our protocol where we found resorption of the MSC deposit after six months in all individuals.

Stem-cell derived exosomes have been shown to play a major role in tissue anti-aging and regeneration [40-42]. We know that exosomes can be used to rewire aging processes in several tissues such as cartilage, myocardium, skin or others [43-45]. The main effect of exosomes are their immunomodulatory effects in general inflammatory responses [46]. This is in accordance with recent exosome investigations and studies focusing on the effects of inflammation on tissue senescence [47-49]. Especially the systemic application of autologous exosomes was source of investigation during the last years. Cho et al. described the effects of exosomes on atopic dermatitis and found alleviation of symptoms after systemic administration [50]. This is also in accordance with the report of Roh YJ, et al. [51]. Sun et al. found MSC derived exosomes to alleviate diabetes mellitus by reversing peripheral insulin resistance and by relieving beta-cell destruction [52]. Zhang et al. highlighted the role of exosomes to promote remyelination and to reduce neuroinflammation in a mouse model [53]. Lee et al. showed exosomes to attenuate inflammatory processes in a model of inflammatory bowel disease [54]. In the current report, we present a useful and safe method to administer a deposit of autologous MSC combined with MSC-derived exosomes within a 3-dimension hyaluronic acid matrix for an intramuscular slow-resorbing depot. We showed significant improvement of systemic inflammation processes and found that the 3D-stem cell deposit was resorbed in all individuals after six months. We further provide reliable data that the deposit can be monitored with ultrasound as described above. We currently are investigating the 3D-deposit in patients with severe osteoarthritis as a possible therapy to relieve symptoms and enhance quality of life.

Further randomized controlled trials are necessary to obtain reliable data on the efficacy of the 3D-stemcell depot in certain medical conditions involving systemic inflammation. We also explicitly highlight the study of Varadhan et al. who pointed out the relevance of pro-inflammatory cytokines on overall mortality in their cohort. This is also in accordance with Puzianowska- Kuznicka et al. who found IL-6 and CRP levels to be good predictors of physical and cognitive performance and the risk of mortality in both the entire elderly population and in successfully aging individuals.

According to the results of our study and according to the fact that such pro-inflammatory parameters significantly decreased after administration of the 3D-stem cell deposit one can extrapolate the results as demonstrated by Varadhan R, et al. [13] and Puzianowska-Kuznicka M, et al. [14] suggesting lower mortality and extended lifespan after 3D-stem cell deposit administration. As a consequence and according to our data, the 3D-stem cell deposit might be an easy, effective and safe longevity concept by immunomodulation and by alleviating chronic inflammation.

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